United States Environmental Criteria and
Environmental Protection Assessment Office
Agency Research Triangle Park, NC 27711
EPA-600/8-83/028dF
June 1986
Research and Development
Air Quality
Criteria for Lead
Volume IV of IV
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EPA/600/8-83/028dF
June 1986
Air Quality Criteria for Lead
Volume IV of IV
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Research Triangle Park, NC 27711
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade
names or commercial products does not constitute endorsement or
recommendation.
ii
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ABSTRACT
The document evaluates and assesses scientific information on the health
and welfare effects associated with exposure to various concentrations of lead
in ambient air. The literature through 1985 has been reviewed thoroughly for
information relevant to air quality criteria, although the document is not
intended as a complete and detailed review of all literature pertaining to
lead. An attempt has been made to identify the major discrepancies in our
current knowledge and understanding of the effects of these pollutants.
Although this document is principally concerned with the health and
welfare effects of lead, other scientific data are presented and evaluated in
order to provide a better understanding of this pollutant in the environment.
To this end, the document includes chapters that discuss the chemistry and
physics of the pollutant; analytical techniques; sources, and types of
emissions; environmental concentrations and exposure levels; atmospheric
chemistry and dispersion modeling; effects on vegetation; and respiratory,
physiological, toxicological, clinical, and epidemiological aspects of human
exposure.
iii
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CONTENTS
Page
VOLUME I
Chapter 1. Executive Summary and Conclusions 1-1
VOLUME II
Chapter 2. Introduction 2-1
Chapter 3. Chemical and Physical Properties 3-1
Chapter 4. Sampling and Analytical Methods for Environmental Lead 4-1
Chapter 5. Sources and Emissions 5-1
Chapter 6. Transport and Transformation 6-1
Chapter 7. Environmental Concentrations and Potential Pathways to Human Exposure .. 7-1
Chapter 8. Effects of Lead on Ecosystems 8-1
VOLUME III
Chapter 9. Quantitative Evaluation of Lead and Biochemical Indices of Lead
Exposure in Physiological Media 9~*
Chapter 10. Metabolism of Lead 10~l
Chapter 11. Assessment of Lead Exposures and Absorption in Human Populations 11~1
Volume IV
Chapter 12. Biological Effects of Lead Exposure 1Z~1
Chapter 13. Evaluation of Human Health Risk Associated with Exposure to Lead
and Its Compounds 13
iv
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TABLE OF CONTENTS
Page
LIST OF FIGURES ix
LIST OF TABLES ix
12. BIOLOGICAL EFFECTS OF LEAD EXPOSURE 12-1
12.1 INTRODUCTION 12-1
12.2 SUBCELLULAR EFFECTS OF LEAD IN HUMANS AND EXPERIMENTAL ANIMALS 12-3
12.2.1 Effects of Lead on the Mitochondrion 12-4
12.2.1.1 Effects of Lead on Mitochondrial Structure 12-4
12.2.1.2 Effects of Lead on Mitochondrial Function 12-5
12.2.1.3 In Vivo Studies 12-5
12.2.1.4 In Vitro Studies 12-7
12.2.2 Effects of Lead on the Nucleus 12-8
12.2.3 Effects of Lead on Membranes 12-9
12.2.4 Other Organellar Effects of Lead 12-10
12.2.5 Summary of Subcellular Effects of Lead 12-10
12.3 EFFECTS OF LEAD ON HEME BIOSYNTHESIS AND ERYTHROPOIESIS/ERYTHROCYTE
PHYSIOLOGY IN HUMANS AND ANIMALS 12-13
12.3.1 Effects of Lead on Heme Biosynthesis 12-13
12.3.1.1 Effects of Lead on 6-Aminolevulinic Acid Synthetase 12-14
12.3.1.2 Effects of Lead on 6-Aminolevulinic Acid Dehydrase and
ALA Accumulation/Excretion 12-15
12.3.1.3 Effects of Lead on Heme Formation from Protoporphyrin ... 12-20
12.3.1.4 Effects of Lead on Coproporhyrin 12-27
12.3.2 Effects of Lead on Erythropoiesis and Erythrocyte Physiology 12-28
12.3.2.1 Effects of Lead on Hemoglobin Production 12-28
12.3.2.2 Effects of Lead on Erythrocyte Morphology and Survival .. 12-29
12.3.2.3 Effects of Lead on Pyrimidine-S'-Nucleotidase Activity
and Erythropoietic Pyrimidine Metabolism 12-31
12.3.3 Effects of Alkyl Lead on Heme Synthesis and Erythopoiesis 12-33
12.3.4 The Interrelationship of Lead Effects on Heme Synthesis and
the Nervous System 12-34
12.3.5 Interference with Vitamin D Metabolism and Associated
Physiological Processes 12-37
12.3.5.1 Relevant Clinical Studies 12-38
12.3.5.2 Experimental Studies 12-39
12.3.5.3 Implications of Lead Effects on Vitamin D Metabolism 12-40
12.3.6 Summary and Overview 12-43
12.3.6.1 Lead Effects on Heme Biosynthesis 12-43
12.3.6.2 Lead Effects on Erythropoiesis and
Erythrocyte Physiology 12-48
12.3.6.3 Effects of Lead on Erythropoietic Pyrimidine
Metabol i sm 12-48
12.3.6.4 Effects of Alkyl Lead Compounds on Heme Biosynthesis
and Erythropoiesis 12-49
12.3.6.5 Relationships of Lead Effects on
Heme Synthesis and Neurotoxicity 12-49
12.3.6.6 Summary of Effects of Lead on Vitamin D Metabolism 12-50
12.4 NEUROTOXIC EFFECTS OF LEAD 12-52
12.4.1 Introduction 12-52
12.4.2 Human Studies 12-53
12.A.2.1 Neurotoxic Effects of Lead Exposure in Adults 12-56
12.4.2.2 Neurotoxic Effects of Lead Exposure in Children 12-68
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TABLE OF CONTENTS (continued).
12.4.3 Animal Studies 12-110
12.4.3.1 Behavioral Toxicity: Critical Periods for Exposure and
Expression of Effects 12-112
12.4.3.2 Morphological Effects 12-139
12.4.3.3 Electrophysiological Effects 12-142
12.4.3.4 Biochemical Alterations 12-145
12.4.3.5 Accumulation and Retention of Lead in the Brain 12-150
12.4.4 Integrative Summary of Human and Animal Studies of Neurotoxicity .. 12-155
12.4.4.1 Internal Exposure Levels at Which Adverse
Neurobehavioral Effects Occur 12-155
12.4.4.2 The Question of Irreversibility 12-158
12.4.4.3 Early Development and the Susceptibility to
Neural Damage 12-158
12.4.4.4 Utility of Animal Studies in Drawing Parallels
to the Human Condition 12-159
12.5 EFFECTS OF LEAD ON THE KIDNEY 12-164
12.5.1 Historical Aspects 12-164
12.5.2 Lead Nephropathy in Childhood 12-164
12.5.3 Lead Nephropathy in Adults 12-165
12.5.3.1 Lead Nephropathy Following Childhood Lead Poisoning 12-166
12.5.3.2 "Moonshine" Lead Nephropathy 12-167
12.5.3.3 Occupational Lead Nephropathy 12-167
12.5.3.4 Lead and Gouty Nephropathy 12-172
12.5.3.5 Lead and Hypertensive Nephrosclerosis 12-175
12.5.3.6 General Population Studies 12-177
12.5.4 Mortality Data 12-178
12.5.5 Experimental Animal Studies of the Pathophysiology of
Lead Nephropathy 12-179
12.5.5.1 Lead Uptake By the Kidney 12-179
12.5.5.2 Intracellular Binding of Lead in the Kidney 12-180
12.5.5.3 Pathological Features of Lead Nephropathy 12-181
12.5.5.4 Functional Studies 12-181
12.5.6 Experimental Studies of the Biochemical Aspects of
Lead Nephrotoxicity 12-183
12.5.6.1 Membrane Marker Enzymes and Transport Functions 12-183
12.5.6.2 Mitochondrial Respiration/Energy-Linked
Transformation 12-184
12.5.6.3 Renal Heme Biosynthesis 12-185
12.5.6.4 Alteration of Renal Nucleic Acid/Protein Synthesis 12-187
12.5.6.5 Lead Effects on the Renin-Angiotension System 12-188
12.5.6.6 Effects of Lead on Uric Acid Metabolism 12-189
12.5.6.7 Effects of Lead on Kidney Vitamin D Metabolism 12-189
12.5.7 General Summary: Comparison of Lead's Effects on Kidneys in
Humans and Animal Models 12-190
12.6 EFFECTS OF LEAD ON REPRODUCTION AND DEVELOPMENT 12-192
12.6.1 Human Studies 12-192
12.6.1.1 Historical Evidence 12-192
12.6.1.2 Effects of Lead Exposure on Reproduction 12-193
12.6.1.3 Placental Transfer of Lead 12~a7
12.6.1.4 Effects of Lead on the Developing Human 12no
12.6.1.5 Summary of the Human Data 12-202
vi
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TABLE OF CONTENTS (continued).
12.6.2 Animal Studies 12-202
12.6.2.1 Effects of Lead on Reproduction 12-202
12.6.2.2 Effects of Lead on the Offspring 12-206
12.6.2.3 Effects of Lead on Avian Species 12-219
12.6.3 Summary 12-219
12.7 GENETOXIC AND CARCINOGENIC EFFECTS OF LEAD '.'.'.'.'.'. 12-221
12.7.1 Introduction 12-221
12.7.2 Carcinogenesis Studies with Lead and its Compounds 12-224
12.7.2.1 Human Epidemic!ogical Studies 12-224
12.7.2.2 Induction of Tumors in Experimental Animals 12-229
12.7.2.3 Cell Transformation 12-234
12.7.3 Genotoxicity of Lead 12-236
12.7.3.1 Chromosomal Aberrations 12-236
12.7.3.2 Sister Chromatid Exchange 12-240
12.7.3.3 Effect of Lead on Bacterial and Mammalian
Mutagenesis Systems 12-242
12.7.3.4 Effect of Lead on Parameters of DNA
Structure and Functi on 12-242
12.7.4 Lead as an Initiator and Promoter of Carcinogenesis 12-244
12.7.5 Summary and Conclusions 12-244
12.8 EFFECTS OF LEAD ON THE IMMUNE SYSTEM 12-246
12.8.1 Development and Organization of the Immune System 12-246
12.8.2 Host Resistance 12-247
12.8.2.1 Infectivity Models 12-248
12.8.2.2 Tumor Models and Neoplasia 12-250
12.8.3 Humoral Immunity 12-251
12.8.3.1 Antibody Titers 12-251
12.8.3.2 Enumeration of Antibody Producing Cells
(Plaque-Forming Cells) 12-252
12.8.4 Cell-Mediated Immunity 12-254
12.8.4.1 Delayed-Type Hypersensitivity 12-254
12.8.4.3 Interferon 12-256
12.8.5 Lymphocyte Activation by Mitogens 12-256
12.8.5.1 In Vivo Exposure 12-256
12.8.5.2 Tn Vvtro Exposure 12-258
12.8.6 MacrophageTunction 12-259
12.8.7 Mechanisms of Lead Immunomodulation 12-261
12.8.8 Summary 12-261
12.9 EFFECTS OF LEAD ON OTHER ORGAN SYSTEMS 12-262
12.9.1 The Cardiovascular System 12-262
12.9.2 The Hepatic System 12-264
12.9.3 The Gastrointestinal System 12-266
12.9.4 The Endocrine System 12-268
12.10 CHAPTER SUMMARY 12-271
12.10.1 Introduction 12-271
12.10.2 Subcel1ular Effects of Lead 12-271
12.10.3 Effects of Lead on Heme Biosynthesis, Erythropoiesis, and
Erythrocyte Physiology in Humans and Animals 12-274
12.10.4 Neurotoxic Effects of Lead 12-281
12.10,4.1 Internal Exposure Levels at Which Adverse
Neurobehavioral Effects Occur 12-281
vi i
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TABLE OF CONTENTS (continued).
12.10.4.2 The Question of Irreversibility 12-283
12.10.4.3 Early Development and the Susceptibility to Neural
Damage 12-283
12.10.4.4 Utility of Animal Studies in Drawing Parallels to the
Human Condition 12-284
12.10.5 Effects of Lead on the Kidney 12-286
12.10.6 Effects of Lead on Reproduction and Development 12-287
12.10.7 Genotoxic and Carcinogenic Effects of Lead 12-289
12.10.8 Effects of Lead on the Immune System 12-289
12.10.9 Effects of Lead on Other Organ Systems 12-289
12.11 REFERENCES 12-291
APPENDIX 12-A 12A-1
13.1 INTRODUCTION 13-1
13.2 EXPOSURE ASPECTS 13-2
13.2.1 Sources of Lead Emission in the United States 13-2
13.2.2 Environmental Cycling of Lead 13-4
13.2.3 Levels of Lead in Various Media of Relevance to Human Exposure 13-5
13.2.3.1 Ambient Air Lead Levels 13-6
13.2.3.2 Levels of Lead in Dust 13-6
13.2.3.3 Levels of Lead in Food 13-7
13.2.3.4 Lead Levels in Drinking Water 13-7
13.2.3.5 Lead in Other Media 13-11
13.2.3.6 Cumulative Human Lead Intake From Various Sources 13-11
13.3 LEAD METABOLISM: KEY ISSUES FOR HUMAN HEALTH RISK EVALUATION 13-11
13.3.1 Differential Internal Lead Exposure Within Population Groups 13-12
13.3.2 Indices of Internal Lead Exposure and Their Relationship to External
Lead Levels and Tissue Burdens/Effects 13-13
13.4 DEMOGRAPHIC CORRELATES OF HUMAN LEAD EXPOSURE AND RELATIONSHIPS BETWEEN
EXTERNAL AND INTERNAL LEAD EXPOSURE INDICES 13-17
13.4.1 Demographic Correlates of Lead Exposure 13-17
13.4.2 Relationships Between External and Internal Lead Exposure Indices 13-19
13.4.3 Proportional Contributions of Lead in Various Media to Blood Lead in
Human Populations 13-26
13.5 BIOLOGICAL EFFECTS OF LEAD RELEVANT TO THE GENERAL HUMAN POPULATION 13-27
13.5.1 Introduction 13-27
13.5.2 Dose-Effect Relationship for Lead-Induced Health Effects 13-32
13.5.2.1 Human Adults 13-32
13.5.2.2 Children 13-34
13.6 DOSE-RESPONSE RELATIONSHIPS FOR LEAD IN HUMAN POPULATIONS 13-41
13.7 POPULATIONS AT RISK 13-44
13.7.1 Children as a Population at Risk 13-44
13.7.1.1 Inherent Susceptibility of the Young 13-45
13.7.1.2 Exposure Consideration 13-45
13.7.2 Pregnant Women and the Conceptus as a Population at Risk 13-46
13.7.3 Middle-Aged White Males (Aged 40-59) as a Population at Risk 13-47
13.7.4 Description of the United States Population in Relation to Potential
Lead Exposure Risk 13-47
13.8 SUMMARY AND CONCLUSIONS 13-49
13.9 REFERENCES 13-51
viii
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LIST OF FIGURES
Figure Page
12-1 Effects on lead (Pb) on heme biosynthesis 12-14
12-2 Regression of IQ scores against blood lead levels, with 95% confidence
band. Double values indicated by triangle 12-93
12-3 (a) Predicted SW voltage and 95% confidence bounds in 13- and 75-month-old
children as a function of blood lead level, (b) Scatter plots of SW data
from children aged 13-47 months with predicted regression lines for ages
18, 30, and 42 months, (c) Scatter plots for children aged 48-75 months
with predicted regression lines for ages 54 and 66 months. These graphs
depict the linear interaction of blood lead level and age 12-105
12-4 Peroneal nerve conduction velocity versus blood lead level, for children
living in a smelter area of Idaho, 1974 12-109
13-1 Pathways of lead from the environment to man, main compartments involved
in partitioning of internal body burden of absorbed/retained lead, and
main routes of lead excretion 13-3
13-2 Body compartments involved in partitioning, retention, and excretion of
absorbed lead and selected target organs for lead toxicity 13-15
13-3 Geometric mean blood lead levels by race and age for younger children in the
NHANES II study, the Kellogg/Silver Valley, and New York childhood screening
studies 13-18
13-4 Multi-organ impact of reduction of heme body pool by lead 13-31
13-5 Dose-response for elevation of EP as a function of blood lead level using
probit analysis 13-42
13-6 Dose-response curve for FEP as a function of blood lead level:
in subpopulations 13-43
13-7 EPA calculated dose-response curve for ALA-U 13-43
LIST OF TABLES
Table
12-1 Summary of studies on nerve conduction velocity in groups of lead-exposed
subjects
12-2 Summary of studies on neurobehavioral functions of lead-exposed children ..
12-3 Effects of lead on activity in rats and mice
12-4 Recent animal toxicology studies of lead's effects on learning in rodents .
12-5 Recent animal toxicology studies of lead's effects on learning in primates
12-6 Summary of key studies of morphological effects of vn vivo lead exposure ..
12-7 Summary of key studies of electrophysiological effects of HI vivo lead
exposure
12-8 Summary of key studies on biochemical effects of ijn vivo lead exposure
12-9 Index of blood lead and brain lead levels following exposure
12-10 Summary of key studies of iji vitro lead exposure
12-11 Morphological features of lead nephropathy in various species
12-12 Effects of lead exposure on aspects of renal heme biosynthesis
12-13 Statistics on the effect of lead on pregnancy
12-14 Effects of prenatal exposure to lead on the offspring of laboratory and
domestic animals: studies using oral or inhalation routes of exposure
Page
12-62
12-73
12-116
12-118
12-128
12-140
12-143
12-146
12-151
12-162
12-182
12-186
12-193
12-207
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LIST OF TABLES (continued).
Table Page
12-15 Effects of prenatal lead exposure on offspring of laboratory animals:
results of studies employing administration of lead by injection 12-209
12-16 Reproductive performance of Fx lead-intoxicated rats (means ± standard
errors) 12-212
12-17 Expected and observed deaths and standardized mortality ratios for
malignant neoplasms Jan. 1, 1947 - Dec. 31, 1979 for lead smelter and
battery pi ant workers 12-225
12-18 Expected and observed deaths resulting from specified malignant neoplasms
for lead smelter and battery plant workers and levels of significance by
type of statistical analysis according to one-tailed tests 12-226
12-19 Examples of studies on the incidence of tumors in experimental animals
exposed to lead compounds 12-230
12-20 Mortality and kidney tumors in rats fed lead acetate for two years 12-234
12-21 Cytogenetic investigations of cells from individuals exposed to lead:
positive studies 12-237
12-22 Cytogenetic investigations of cells from individuals exposed to lead:
negative studies 12-238
12-23 Effect of lead on host resistance to infectious agents 12-248
12-24 Effect of lead on antibody titers 12-251
12-25 Effect of lead on the development of antibody-producing cells 12-253
12-26 Effect of lead on cell-mediated immunity 12-255
12-27 Effect of lead exposure on mitogen activation of lymphocytes 12-257
12-28 Effect of lead on macrophage and reticyloendothelial system function 12-260
12-A Tests commonly used in a psycho-educational battery for children 12-A2
13-1 Summary of baseline human exposures to lead 13-8
13-2 Relative baseline human lead exposures expressed per kilogram body weight 13-9
13-3 Summary of potential additive exposures to lead (ug/day) 13-10
13-4 Summary of blood inhalation slopes, O) 13-20
13-5 Estimated contribution of leaded gasoline to blood lead by inhalation and
non-inhalation pathways 13-25
13-6 Contributions from various media to blood lead (ng/dl) of U.S. children
(Age = 2 years): Background levels and incremental contributions from air 13-28
13-7 Summary of lowest observed effect levels for key lead-induced health effects
in adults 13-33
13-8 Summary of lowest observed effect levels for key lead-induced health effects
in children 13-35
13-9 EPA-estimated percentage of subjects with ALA-U exceeding limits for various
blood lead levels 13-44
13-10 Provisional estimate of the number of individuals in urban and rural
population segments at greatest potential risk to lead exposure 13-48
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LIST OF ABBREVIATIONS
AAS
Ach
ACTH
ADCC
ADP/0 ratio
AIDS
AIHA
All
ALA
ALA-D
ALA-S
ALA-U
APDC
APHA
ASTM
ASV
ATP
B-cells
Ba
BAL
BAP
BSA
BUN
BW
C.V.
CaBP
CaEDTA
CaNa2EDTA
CBD
Cd
CDC
CEC
CEH
CFR
CMP
CNS
CO
COHb
CP-U
cBah
D.F.
DA
6-ALA
DCMU
DPP
DMA
DTH
EEC
EEG
EMC
EP
Atomic absorption spectrometry
Acetylcholine
Adrenocorticotrophic hormone
Antibody-dependent cell-mediated cytotoxicity
Adenosine diphosphate/oxygen ratio
Acquired immune deficiency syndrome
American Industrial Hygiene Association
Angiotensin II
Aminolevulinic acid
Aminolevulinic acid dehydrase
Aminolevulinic acid synthetase
Aminolevulinic acid in urine
Ammonium pyrrolidine-dithiocarbamate
American Public Health Association
Amercian Society for Testing and Materials
Anodic stripping voltammetry
Adenosine triphpsphate
Bone marrow-derived lymphocytes
Barium
British anti-Lewisite (dimercaprol)
benzo(a)pyrene
Bovine serum albumin
Blood serum urea nitrogen
Body weight
Coefficient of variation
Calcium binding protein
Calci urn ethylenedi ami netetraacetate
Calcium disodium ethylenediaminetetraacetate
Central business district
Cadmium
Centers for Disease Control
Cation exchange capacity
Center for Environmental Health
reference method
Cytidine monophosphate
Central nervous system
Carbon monoxide
Carboxyhemoglobi n
Urinary coproporphyrin
plasma clearance of p-aminohippuric acid
Copper
Degrees of freedom
Dopamine
delta-aminolevulinic acid
[3-(3,4-dichlorophenyl)-l,l-dimethyl urea
Differential pulse polarography
Deoxyribonucleic acid
Delayed-type hypersensitivity
European Economic Community
Electroencephalogram
Encephalomyocardi ti s
Erythrocyte protoporphyrin
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LIST OF ABBREVIATIONS (continued).
EPA U.S. Environmental Protection Agency
FA Fulvic acid
FDA Food and Drug Administration
Fe Iron
FEP Free erythrocyte protoporphyrin
FY Fiscal year
G.M. Grand mean
G-6-PD Glucose-6-phosphate dehydrogenase
GABA Gamma-aminobutyric acid
GALT Gut-associated lymphoid tissue
GC Gas chromatography
GFR Glomerular filtration rate
HA Humic acid
Hg Mercury
hi-vol High-volume air sampler
HPLC High-performance liquid chromatography
i-ii. Intramuscular (method of injection)
i-P- Intraperitoneally (method of injection)
i-V. Intravenously (method of injection)
IAA Indol-3-ylacetic acid
IARC International Agency for Research on Cancer
ICD International classification of diseases
ICP Inductively coupled plasma emission spectroscopy
IDMS Isotope dilution mass spectrometry
IF Interferon
HE Isotopic Lead Experiment (Italy)
IRPC International Radiological Protection Commission
K Potassium
LDH-X Lactate dehydrogenase isoenzyme x
LCj-Q Lethyl concentration (50 percent)
LDgQ Lethal dose (50 percent)
LH Luteinizing hormone
LIPO Laboratory Improvement Program Office
In Natural logarithm
LPS Lipopolysaccharide
LRT Long range transport
mRNA Messenger ribonucleic acid
ME Mercaptoethanol
MEPP Miniature end-plate potential
MES Maximal electroshock seizure*
MeV Mega-electron volts
MLC Mixed lymphocyte culture
HMD Mass median diameter
MMAD Mass median aerodynamic diameter
Mn Manganese
MND Motor neuron disease
MSV Moloney sarcoma virus
MTD Maximum tolerated dose
n Number of subjects or observations
N/A Not Available
XI1
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LIST OF ABBREVIATIONS
NA
NAAQS
NAD
NADB
NANS
NAS
NASN
NBS
NE
NFAN
NFR-82
NHANES II
Ni
NTA
OSHA
P
P
PAH
Pb
PBA
Pb(Ac),
PbB *
PbBrCl
PBG
PFC
pH
PHA
PHZ
PIXE
PMN
PNO
PNS
p.o.
ppm
PRA
PRS
PWM
Py-5-N
RBC
RBF
RCR
redox
RES
RLV
RNA
S-HT
SA-7
s.c.
scm
S.D.
SOS
S.E.M.
Not Applicable
National ambient air quality standards
Nicotinamide Adenine Dinucleotide
National Aerometric Data Bank
National Air Monitoring Station
National Academy of Sciences
National Air Surveillance Network
National Bureau of Standards
Norepinephrine
National Filter Analysis Network
Nutrition Foundation Report of 1982
National Health Assessment and Nutritional Evaluation Survey II
Nickel
Nitrilotriacetonitrile
Occupational Safety and Health Administration
Phosphorus
Significance symbol
Para-aminohippuric acid
Lead
Air lead
Lead acetate
concentration of lead in blood
Lead (II) bromochloride
Porphobilinogen
Plaque-forming cells
Measure of acidity
Phytohemaggluti ni n
Polyacrylamide-hydrous-zirconia
Proton-induced X-ray emissions
Polymorphonuclear leukocytes
Post-natal day
Peripheral nervous system
Per os (orally)
Parts per million
Plasma renin activity
Plasma renin substrate
Pokeweed mitogen
Pyri mi de-5'-nucleoti dase
Red blood cell; erythrocyte
Renal blood flow
Respiratory control ratios/rates
Oxidation-reduction potential
Reticuloendothelial system
Rauscher leukemia virus
Ribonucleic acid
Serotonin
Simian adenovirus
Subcutaneously (method of injection)
Standard cubic meter
Standard deviation
Sodium dodecyl sulfate
Standard error of the mean
xiii
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LIST OF ABBREVIATIONS (continued).
SES
SCOT
slg
SLAMS
SMR
Sr
SRBC
SRMs
STEL
SW voltage
T-cells
t-tests
TBL
TEA
TEL
TIBC
TML
TMLC
TSH
TSP -
U.K.
UMP
USPHS
VA
WHO
XRF
X^
Zn
ZPP
Socioeconomic status
Serum glutamic oxaloacetic transaminase
Surface immunoglobulin
State and local air monitoring stations
Standardized mortality ratio
Strontium
Sheep red blood cells
Standard reference materials
Short-term exposure limit
Slow-wave voltage
Thymus-derived lymphocytes
Tests of significance
Tri-n-butyl lead
Tetraethyl-ammoni urn
Tetraethyllead
Total iron binding capacity
Tetramethyllead
Tetramethyllead chloride
Thyroid-stimulating hormone
Total suspended particulate
United Kingdom
Uridine monophosphate
U.S. Public Health Service
Veterans Administration
Deposition velocity
Visual evoked response
World Health Organization
X-Ray fluorescence
Chi squared
Zinc
Erythrocyte zinc protoporphyrin
MEASUREMENT ABBREVIATIONS
dl
ft
9,
g/gal
g/ha-mo
km/hr
1/min
mg/km
ug/m3
mm
urn
(jmol
ng/cm2
nm
nM
sec
t
deciliter
feet
gram
gram/gallon
gram/hectare-month
kilometer/hour
1i ter/mi nute
milli gram/ki1ometer
microgram/cubic meter
millimeter
micrometer
micromole
nanograms/square centimeter
nanometer
nanomole
second
tons
xiv
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GLOSSARY VOLUME IV
ADP/0 ratio - a measure of the rate of respiration; the ratio of adenosine
diphosphate concentration to oxygen levels increases as
respiration is impaired
active transport - the translocation of a solute across a membrane by means of
an energy-dependent carrier system capable of moving against
a concentration gradient
affective function - pertaining to emotion
asthenospermia - loss or reduction of the motility of spermatozoa
azotemia - an excess of urea and other nitrogenous compounds in the blood
basal ganglia - all of the large masses of gray matter at the base of the
cerebral hemispheres, including the corpus striatum, subthalamic
nucleus, and substantia nigra
basophilic stippling - a histochemical appearance characteristic of immature
erythrocytes
cognitive function - pertaining to reasoning, judging, conceiving, etc.
corpuscular volume - red blood cell volume
cristae - shelf-like infoldings of the inner membrane of mitochondria
cytomegaly - markedly enlarged cells
demyelination - destruction of the protective myelin sheath which surrounds
most nerves
depolarization - the electrophysiological process underlying neural transmission
desaturation kinetic study - a form of kinetic study in which the rate of release
of a species from its binding is studied
desquamation - shedding, peeling, or scaling off
disinhibition - removal of a tonic inhibitory effect
endoneurium - the delicate connective tissue enveloping individual nerve fibers
within a nerve
erythrocyte - red blood cell
erythropoiesis - the formation of red blood cells
feedback derepression - the deactivation of a represser
hepatocyte - a parenchyma! liver cell
xv
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hyalinization - a histochemical marker characteristic of degeneration
hyperkalemia - a greater than normal concentration of potassium ions in the
circulating blood
hyperplasia - increased numbers of cells
hypertrophy - increased size of cells
hypochromic - containing less than the normal amount of pigment
hyporeninemic hypoaldosteronism - pertaining to a systemic deficiency of renin
and aldosterone
inclusion bodies - any foreign substance contained or entrapped within a cell
isocortex - cerebral cortex
lysosomes - a cytoplasmic, membrane-bound particle containing hydrolyzing
enzymes
macrophage - large scavenger cell that ingests bacteria, foreign bodies, etc.
(Na+, K+)-ATPase - an energy-dependent enzyme which transports sodium and
potassium across cell membranes
natriuresis - enhanced urinary excretion of sodium
normocytic - refers to normal, heal thy-looking erythrocytes
organotypic - disease or cell mixture representative of a specific organ
oxidative phosphorylation - the generation of cellular energy in the presence
of oxygen
paresis - partial or incomplete paralysis
pathognomic feature - characteristic or indicative of a disease
polymorphonuclear leukocytes - leukocytes (white blood cells) having nuclei of
various forms
respiratory control rates (RCRs) - measure of intermediary metabolism
reticulocytosis - an increase in the number of circulating immature red blood
cells
synaptogenesis - the formation of neural connections (synapses)
synaptosomes - morphological unit composed of nerve terminals and the attached
synapse
teratogenic - affecting the development of an organism
teratospermia - a condition characterized by the presence of malformed
spermatozoa
xv i
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Chapter 12: Biological Effects of Lead Exposure
Contributing Authors
Dr. Max Costa
Department of Pharmacology
University of Texas Medical School
Houston, TX 77025
Dr. J. Michael Davis
Environmental Criteria and Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Jack Dean
Immunobiology Program and Immunotoxicology/
Cell Biology Program
CUT
P.O. Box 12137
Research Triangle Park, NC 27709
Dr. Bruce Fowler
Laboratory of Pharmacology
NIEHS
P.O. Box 12233
Research Triangle Park, NC 27709
Dr. Lester Grant
Director, Environmental Criteria and
Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Ronald D. Hood
Department of Biology
The University of Alabama
P.O. Box 1927
University, AL 35486
Dr. Loren Keller
School of Veterinary Medicine
University of Idaho
Moscow, ID 83843
Dr. David Lawrence
Microbiology and Immunology Department
Albany Medical College of Union University
Albany, NY 12208
Dr. Paul Mushak
Department of Pathology
UNC School of Medicine
Chapel Hill, NC 27514
Dr. David Otto
Clinical Studies Division
MD-58
U.S. Environmental Protection
Agency
Research Triangle Park, NC 27711
Dr. Magnus Piscator
Department of Environmental Hygiene
The Karolinska Institute 104 01
Stockholm
Sweden
Dr. John F. Rosen
Department of Pediatrics
Montefiore Hospital and
Medical Center
New York, NY 10467
Dr. Stephen R. Schroeder
Division for Disorders of
Development and Learning
Biological Sciences Research Center
University of North Carolina
Chapel Hill, NC 27514
Dr. Richard P. Wedeen
V.A. Medical Center
Tremont Avenue
East Orange, NJ 07019
Dr. David Weil
Environmental Criteria and
Assessment Office
MD-52
U.S. Environmental Protection
Agency
Research Triangle Park, NC 27711
xvii
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The following persons reviewed this chapter at EPA's request. The evaluations
and conclusions contained herein, however, are not necessarily those of the
reviewers.
Dr. Carol Angle
Department of Pediatrics
University of Nebraska
College of Medicine
Omaha, NE 68105
Dr. Lee Annest
Division of Health Examin. Statistics
National Center for Health Statistics
3700 East-West Highway
Hyattsville, MD 20782
Dr. Donald Barltrop
Department of Child Health
Westminister Children's Hospital
London SW1P 2NS
England
Dr. Irv Billick
Gas Research Institute
8600 West Bryn Mawr Avenue
Chicago, IL 60631
Dr. Joe Boone
Clinical Chemistry and
Toxicology Section
Center for Disease Control
Atlanta, GA 30333
Dr. Robert Bornschein
University of Cincinnati
Kettering Laboratory
Cincinnati, OH 45267
Dr. A. C. Chamberlain
Environmental and Medical Sciences Division
Atomic Energy Research Establishment
Harwell 0X11
England
Dr. Neil Chernoff
Division of Developmental Biology
MD-67
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Julian Chisolm
Baltimore City Hospital
4940 Eastern Avenue
Baltimore, MD 21224
Dr. Jerry Cole
International Lead-Zinc Research
Organization
292 Madison Avenue
New York, NY 10017
Dr. Anita Curran
Commissioner of Health
Westchester County
White Plains, NY 10607
Dr. Cliff Davidson
Department of Civil Engineering
Carnegie-Mellon University
Schenley Park
Pittsburgh, PA 15213
Dr. H. T. Delves
Chemical Pathology and Human
Metabolism
Southampton General Hospital
Southampton S09 4XY
England
Dr. Fred deSerres
Associate Director for Genetics
NIEHS
P.O. Box 12233
Research Triangle Park, NC 27709
Dr. Joseph A. DiPaolo
Laboratory of Biology, Division
of Cancer Cause and Prevention
National Cancer Institute
Bethesda, MD 20205
Dr. Robert Dixon
Laboratory of Reproductive and
Developmental Toxicology
NIEHS
P.O. Box 12233
Research Triangle Park, NC 27711
xvm
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Dr. Clair Ernhart
Department of Psychiatry
Cleveland Metropolitan General Hospital
3395 Scranton Road
Cleveland, OH 44109
Dr. Sergio Fachetti
Section Head - Isotope Analysis
Chemistry Division
Joint Research Center
121020 Ispra
Varese, Italy
Dr. Virgil Perm
Department of Anatomy and Cytology
Dartmouth Medical School
Hanover, NH 03755
Dr. Alf Fischbein
Environmental Sciences Laboratory
Mt. Sinai School of Medicine
New York, NY 10029
Dr. Jack Fowle
Reproductive Effects Assessment Group
U.S. Environmental Protection Agency
RD-689
Washington, DC 20460
Dr. Bruce Fowler
Laboratory of Pharmocology
NIEHS
P.O. Box 12233
Research Triangle Park, NC 27709
Dr. Warren Galke
Department of Biostatisties and Epidemiology
School of Allied Health
East Carolina University
Greenville, NC 27834
Mr. Eric Goldstein
Natural Resources Defense Council, Inc.
122 E. 42nd Street
New York, NY 10168
Dr. Harvey Gonick
1033 Gayley Avenue
Suite 116
Los Angeles, CA 90024
Dr. Robert Goyer
Deputy Director
NIEHS
P.O. Box 12233
Research Triangle Park, NC 27711
Dr. Philippe Grandjean
Department of Environmental Medicine
Institute of Community Health
Odense University
Denmark
Dr. Stanley Gross
Hazard Evaluation Division
Toxicology Branch
U.S. Environmental Protection
Agency
Washington, DC 20460
Dr. Paul Hammond
University of Cincinnati
Kettering Laboratory
Cincinnati, OH 45267
Dr. Kari Hemminki
Institute of Occupational Health
Tyoterveyslaitos-Haartmaninkatu
1 SF-00290 Helsinki 29
Finland
Dr. V. Houk
Center for Disease Control
1600 Clifton Road, NE
Atlanta, GA 30333
Dr. Carole A. Kimmel
Perinatal and Postnatal Evaluation
Branch
National Center for Toxicological
Research
Jefferson, AR 72079
Dr. Kristal Kostial
Institute for Medical Research
and Occupational Health
YU-4100 Zagreb
Yugoslavia
Dr. Lawrence Kupper
Department of Biostatisties
UNC School of Public Health
Chapel Hill, NC 27514
xix
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Dr. Philip Landrigan
Division of Surveillance,
Hazard Evaluation and Field Studies
Taft Laboratories - NIOSH
Cincinnati, OH 45226
Dr. Alais-Yves Leonard
Centre D1Etude De L'Energie Nucleaire
B-2400 Mol
Belgium
Dr. Jane Lin-Fu
Office of Maternal and Child Health
Department of Health and Human Services
Rockville, MD 20857
Dr. Don Lynam
Air Conservation
Ethyl Corporation
451 Florida Boulevard
Baton Rouge, LA 70801
Dr. Kathryn Mahaffey
Division of Nutrition
Food and Drug Administration
1090 Tusculum Avenue
Cincinnati, OH 45226
Dr. Ed McCabe
Department of Pediatrics
University of Wisconsin
Madison, WI 53706
Dr. Chuck Nauman
Exposure Assessment Group
U.S. Environmental Protection Agnecy
Washington, DC 20460
Dr. Herbert L. Needleman
Children's Hospital of Pittsburgh
Pittsburgh, PA 15213
Dr. Forrest H. Nielsen
Grand Forks Human Nutrition Research Center
USDA
Grand Forks, ND 58202
Dr. Stephen Overman
Toxicology Institute
New York State Department of
Health
Empire State Plaza
Albany, NY 12001
Dr. H. Mitchell Perry
V.A. Medical Center
St. Louis, MO 63131
Dr. Jack Pierrard
E.I. duPont de Nemours and
Company, Inc.
Petroleum Laboratory
Wilmington, DE 19898
Dr. Sergio Piomelli
Columbia University Medical School
Division of Pediatric Hematology
and Oncology
New York, NY 10032
Dr. Robert Putnam
International Lead-Zinc
Research Organization
292 Madison Avenue
New York, NY 10017
Dr. Michael Rabinowitz
Children's Hospital Medical Center
300 Longwood Avenue
Boston, MA 02115
Dr. Larry Reiter
Neurotoxicology Division
MD-74B
U.S. Environmental Protection
Agency
Research Triangle Park, NC 27711
Dr. Cecil R. Reynolds
Department of Educational Psychology
Texas A & M University
College Station, TX 77843
Dr. Patricia Rodier
Department of Anatomy
University of Rochester Medical
Center
Rochester, NY 14642
xx
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Dr. Harry Roels
Unite de Toxicologie Industrie!le et Medicale
Um'versite de Louvain
Brussels, Belgium
Dr. John Rosen
Head, Division of Pediatric Metabolism
Montefiore Hospital and Medical Center
111 East 210 Street
Bronx, NY 10467
Dr. Michael Rutter
Department of Psychology
Institute of Psychiatry
DeCrespigny Park
London SE5 SAL
England
Dr. Anna-Maria Seppalainen
Institutes of Occupational Health
Tyoterveyslaitos
Haartmanikatu 1
00290 Helsinki 29
Finland
Dr. Ellen Silbergeld
Environmental Defense Fund
1525 18th Street, NW
Washington, DC 20036
Ms. Marjorie Smith
Department of Psychological Medicine
Hospital for Sick Children
Great Ormond Street
London WC1N 3EM
England
Mr. Peter Harvey
Environment, Health and
Behavior Research Group
59 Selly Wick Road
The University of Birmingham
Birmingham B29 7JF
England
Dr. Ron Snee
E.I. duPont de Nemours and
Company, Inc.
Engineering Department L3167
Wilmington, DE 19898
Dr. F. William Sunderman, Jr.
Department of Pharmacology
University of Connecticut
School of Medicine
Farmington, CT 06032
Dr. Gary Ter Haar
Toxicology and Industrial
Hygiene
Ethyl Corporation
451 Florida Boulevard
Baton Rouge, LA 70801
Dr. Hugh A. Tilson
Laboratory of Behavioral and
Neurological Toxicology
NIEHS
Research Triangle Park, NC 27709
Mr. Ian von Lindern
Department of Chemical Engineering
University of Idaho
Moscow, ID 83843
Dr. William Yule
Institute of Psychiatry
DeCrespigny Park
London SE5 8AF
England
xxi
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Chapter 13: Risk Assessment
Principal Authors
Dr. Lester Grant
Director, Environmental Criteria and
Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Contributing Authors
Dr. Robert Elias
Environmental Criteria and Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Vic Hasselblad
Biometry Division
MD-55
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Dennis Kotchmar
Environmental Criteria and Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Paul Mushak
Department of Pathology
UNC School of Medicine
Chapel Hill, NC 27514
Dr. Alan Marcus
Department of Mathematics
Washington State University
Pullman, Washington 99164-2930
Dr. David Weil
Environmental Criteria and
Assessment Office
U.S Environmental Protection
Agency
Research Triangle Park, NC 27711
xxn
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12. BIOLOGICAL EFFECTS OF LEAD EXPOSURE
12.1 INTRODUCTION
As noted in Chapter 2, air quality criteria documents evaluate scientific knowledge of
relationships between pollutant concentrations and their effects on the environment and public
health. Chapters 3-7 of this document discuss the following: physical and chemical proper-
ties of lead; measurement methods for lead in environmental media; sources of emissions;
transport, transformation, and fate; and ambient concentrations and other aspects of the ex-
posure of the U.S. population to lead in the environment. Chapter 8 evaluates the projected
impact of lead on ecosystems. Chapters 9-11 discuss the following: measurement techniques
for lead in biologic media; aspects related to the uptake, distribution, toxicokinetics, and
excretion of lead; and the relationship of various external and internal lead exposure indices
to each other. This chapter assesses information regarding biological effects of lead expo-
sure, with emphasis on (1) the qualitative characterization of various lead-induced effects
and (2) the delineation of dose-effect relationships for key health effects most likely of
concern at ambient exposure levels currently encountered by the general population of the
United States.
It is clear from the evidence evaluated in this chapter that there exists a continuum of
biological effects associated with lead across a broad range of exposure. At rather low
levels of lead exposure, biochemical changes, such as the disruption of certain enzymatic
activities involved in heme biosynthesis and erythropoietic pyrimidine metabolism, are detec-
table. With increasing lead exposure, there are sequentially more pronounced effects on heme
synthesis and a broadening of lead's effects to additional biochemical and physiological
mechanisms in various tissues, such that progressively more severe disruption of the normal
functioning of many different organ systems becomes apparent. In addition to impairment of
heme biosynthesis, signs of disruption of normal functioning of the erythropoietic and nervous
systems are among the earliest effects observed in response to increasing lead exposure. At
increasingly higher exposure levels, more severe disruption of the erythropoietic and nervous
systems occurs; other organ systems are also affected so as to result in the manifestation of
renal effects, disruption of reproductive functions, impairment of immunological functions,
and many other biological effects. At sufficiently high levels of exposure, the damage to the
nervous system and other effects can be severe enough to result in death or, in some cases of
non-fatal lead poisoning, long-lasting sequelae such as permanent mental retardation.
The etiologies of many of the different types of functional disruption of various mamma-
lian organ systems derive (at least in their earliest stages) from lead's effects on certain
subcellular organelles, which result in biochemical derangements (e.g., disruption of heme
12-1
-------
synthesis processes) common to and affecting many tissues and organ systems. Some major
effects of lead on subcellular organelles common to numerous organ systems in mammalian spe-
cies are discussed below in Section 12.2, with particular emphasis on lead effects on mito-
chondrial functions. Subsequent sections of this chapter discuss biological effects of lead
in terms of various organ or physiological systems affected by the element and its compounds
(except for Section 12.7, which assesses genotoxic and carcinogenic effects of lead). Addi-
tional cellular and subcellular aspects of the biological effects of lead are discussed within
respective sections on particular organ systems.
Sections 12.3 to 12.10 have been sequenced generally according to the degree of known
vulnerability of each system to lead. Major emphasis is placed first on detailed discussion
of the effects of lead on heme synthesis and associated multisystem impacts on several
important physiological processes and organ systems. Effects of lead on the two organ systems
classically considered to be most sensitive to lead (i.e., the hematopoietic and the nervous
systems) are further emphasized in early sections. Subsequent sections then discuss addi-
tional effects of lead on the kidney and on reproduction and development (in view of the
impact of lead on the fetus and pregnant women, as well as its gametotoxic effects). Geno-
toxic effects of lead and evidence for possible carcinogenic effects of lead are then re-
viewed, followed by discussion of the effects of lead on the immune system and, lastly, other
organ systems.
This chapter is subdivided mainly according to organ systems to facilitate presentation
of information. It must be noted, however, that in reality, all systems function in delicate
concert to preserve the physiological integrity of the whole organism and all systems are in-
terdependent. Thus, not only do effects in a critical organ often exert impacts on other
organ systems, but low-level effects that might be construed as unimportant in a single speci-
fic system may be of concern in terms of their cumulative or aggregate impact.
Special emphasis is placed on the discussion of the effects of lead exposure in children.
Children are particularly at risk due to sources of exposure, mode of entry, rate of absorp-
tion and retention, and partitioning of lead in soft and hard tissues. The greater sensitivi-
ty of children to lead toxicity, their inability to recognize symptoms, and their dependence
on parents and health care professionals make them an especially vulnerable population re-
quiring special consideration in developing criteria and standards for lead.
In discussing the biological effects of lead, it is important to note that lead has not
been demonstrated to have any beneficial biological effect in humans. Some recent studies
have raised the possibility that lead could be a nutritionally essential element. The primary
evidence for this view has come from a series of articles by Kirchgessner and Reichlmayr-Lais,
who have reported that rats maintained on a semi-synthetic diet low in lead (either 18 or
45 ppb) over several generations showed reduced growth rates (Reichlmayr-Lais and
Kirchgessner, 1981a), disturbances in hematological indices, tissue iron, and iron absorption
12-2
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(Reichlmayr-Lais and Kirchgessner, 1981b,c,d,e; Kirchgessner and Reichlmayr-Lais, 1981a,b),
and changes in certain enzyme activities and metabolite levels (Reichlmayr-Lais and
Kirchgessner, 1981f; Kirchgessner and Reichlmayr-Lais, 1982). Diets containing 18 ppb lead
were associated with the most pronounced effects on iron metabolism and growth as well as on
enzyme activities and metabolite levels. Animals in the Fj-group maintained on a 45-ppb lead
diet showed moderate changes in some hematological indices.
These studies were reviewed by a committee of independent scientists convened by the U.S.
Environmental Protection Agency (Expert Committee on Trace Metal Essentiality, 1983). The
Committee's conclusions were as follows:
1. The Kirchgessner and Reichlmayr-Lais data furnish evidence that is consistent
with and, in some opinions, indicative of a nutritional essentiality of lead
for rats.
2. The evidence is not sufficient to establish nutritional essentiality of lead
for rats.
3. To address the basic issue of nutritional essentiality of lead, additional evi-
dence needs to be obtained under different conditions in the laboratory of
Kirchgessner and Reichlmayr-Lais, as well as by independent investigators;
additional species should also be examined.
The Committee emphasized the difference that apparently exists between lead con-
centrations that are of concern from a toxicologic viewpoint (e.g., those associated with
effects of the various types discussed in this chapter) and much lower lead levels that might
possibly be of nutritional value. Hence the Committee did not perceive any practical incom-
patibility between (a) efforts to reduce lead in the human environment to concentrations that
are unassociated with toxic effects and (b) efforts to define the potential nutritional
essentiality of lead. The Committee further recognized that current public health concerns
for humans clearly focus on lead toxicity effects.
Finally, the question of lead essentiality is largely moot in the debate over lead as a
public health issue. The extent of permanent and pervasive lead contamination in developed
areas of the world is such that concern will remain with excessive lead exposure and asso-
ciated toxicity in human populations. It is virtually inconceivable that lead deficiency in
human populations would ever arise in the first place.
12.2 SUBCELLULAR EFFECTS OF LEAD IN HUMANS AND EXPERIMENTAL ANIMALS
The biochemical or molecular basis for lead toxicity is the ability of the toxicant, as
a metallic cation, to bind to ligating groups in biomolecular substances crucial to normal
physiological functions, thereby interfering with these functions via such mechanisms as
12-3
-------
competition with native essential metals for binding sites, inhibition of enzyme activity, and
inhibition or other alterations of essential ion transport. The relationship of this basis
for lead toxicity to organ- and organelle-specific effects is modulated by the following:
(1) the inherent stability of such binding sites for lead; (2) the compartmentalization
kinetics governing lead distribution among body compartments, among tissues, and within cells;
and (3) differences in biochemical and physiological organization across tissues and cells due
to their specific function. Given complexities introduced by factors 2 and 3, it is not sur-
prising that no single, unifying mechanism of lead toxicity has been demonstrated to apply
across all tissues and organ systems.
In the 1977 Air Quality Criteria Document for Lead, cellular and subcellular effects of
lead were discussed, including effects on various classes of enzymes. Much of the literature
detailing the effects of lead on enzymes per se has entailed study of relatively pure enzymes
iji vitro in the presence of added lead. This was the case for data discussed in the earlier
document and such information continues to appear in the literature. Much of this material is
of questionable relevance for effects of lead In vivo. On the other hand, lead effects on
certain enzymes or enzyme systems are recognized as integral mechanisms of action underlying
the impact of lead on tissues in vivo and are logically discussed in later sections below on
effects at the organ system level.
This subsection is mainly concerned with organellar effects of lead, especially those
that provide some rationale for lead toxicity at higher levels of biological organization.
While a common mechanism at the subcellular level that would account for all aspects of lead
toxicity has not been identified, one fairly common cellular response to lead is the impair-
ment of mitochondrial structure and function; thus, mitochondrion effects are accorded major
attention here. Lead effects on other organelles have not been as extensively studied as
mitochondrion effects; in some cases, it is not clear how the available information, e.g.,
that on lead-containing nuclear inclusion bodies, relates to organellar dysfunction.
12.2.1 Effects of Lead on the Mitochondrion
The mitochondrion is clearly the target organelle for toxic effects of lead on many tis-
sues, the characteristics of"vulnerability varying somewhat with cell type. Given early re-
cognition of this sensitivity, it is not surprising that an extensive body of i_n vivo and j_n
vitro data has accumulated, which can be characterized as evidence of the following: (1)
structural injury to the mitochondrion; (2) impairment of basic cellular energetics and other
mitochondrial functions; and (3) uptake of lead by mitochondria jji vivo and in vitro.
12.2.1.1 Lead Effects on Mitochondrial Structure. Changes in mitochondrial morphology with
lead exposure have been well documented in humans and experimental animals and, in the case of
the kidney, are a rather early response to such exposure. Earlier studies have been reviewed
by Goyer and Rhyne (1973), followed by later updates by Fowler (1978) and Bull (1980).
12-4
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Chronic oral exposure of adult rats to lead (1 percent lead acetate in diet) results in
structural changes in renal tubule mitochondria, including swelling with distortion or loss of
cristae (Goyer, 1968). Such changes have also been documented in renal biopsy tissue of lead
workers (Wedeen et al., 1975; Biagini et al., 1977) and in tissues other than kidney, i.e.,
heart (Malpass et al., 1971; Moore et al., 1975b), liver (Hoffmann et al., 1972), and the cen-
tral (Press, 1977) and peripheral (Brashear et al., 1978) nervous systems.
While it appears that relatively high-level lead exposures are necessary to detect struc-
tural changes in mitochondria in some animal models (Goyer, 1968; Hoffmann et al., 1972),
changes in rat heart mitochondria have been seen at blood lead levels as low as 42 ug/dl.
Also, in the study of Fowler et al. (1980), swollen mitochondria in renal tubule cells were
seen in rats chronically exposed to lead from gestation to 9 months of age at a dietary lead
dosing level as low as 50 ppm and a median blood lead level of 26 ug/dl (range: 15-41 ug/dl).
Taken collectively, these differences point out relative tissue sensitivity, dosing protocol,
relative sensitivity of the various experimental techniques, and the possible effect of
developmental status (Fowler et al., 1980) as important factors in determining lead exposure
levels at which mitochondria are affected in various tissues.
12.2.1.2 Lead Effects on Mitochondria! Function. Both in vivo and in vitro studies dealing
with such effects of lead as the impact on energy metabolism, intermediary metabolism, and
intracellular ion transport have been carried out in various experimental animal models. Of
particular interest for this section are such effects of lead in the developing versus the
adult animal, given the greater sensitivity of the young organism to lead.
12.2.1.3 In Vivo Studies. Uncoupled energy metabolism, inhibited cellular respiration using
succinate and nicotinamide adenine dinucleotide (NAD)-linked substrates, and altered kinetics
of intracellular calcium have all been documented for animals exposed to lead jn vivo, as
reviewed by Bull (1980).
Adult rat kidney mitochondria, following chronic oral feeding of lead in the diet (1 per-
cent lead acetate, 10 or more weeks) showed a marked sensitivity of the pyruvate-NAD reductase
system (Goyer, 1971), as indicated by impairment of pyruvate-dependent respiration indexed by
AOP/0 ratio and respiratory control rates (RCRs). Succinate-mediated respiration in these
animals, however, was not different from controls. In contrast, Fowler et al. (1980) found in
rats exposed jn utero (dams fed 50 or 250 ppm lead) and for 9 months postnatally (50 or 250
ppm lead in their diet) renal tubule mitochondria that exhibited decreased state 3 respiration
and RCRs for both succinate and pyruvate/malate substrates. This may have been due to longer
exposure to lead or a differential effect of lead exposure during early development.
Intraperitoneal administration of lead to adult rats at doses as low as 12 mg/kg over 14
days was associated with inhibition of potassium-stimulated respiration in cerebral cortex
12-5
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slices with impairment of NADPH (NAD phosphate, reduced) oxidation using glucose, but not
pyruvate, as substrate (Bull et al., 1975). This effect occurred at a corresponding blood
lead of 72 ug/dl and a brain lead content of 0.4 ug/g, values below those associated with
overt neurotoxicity. Bull (1977), in a later study, demonstrated that the respiratory re-
sponse of cerebral cortical tissue from lead-dosed rats receiving a total of 60 mg/kg (10
mg/kg x 6 dosings) over 14 days was associated with a marked decrease in turnover of intra-
cellular calcium in a cellular compartment that appears to be the mitochondrion. This is
consistent with the observation of Bouldin et al. (1975) that lead treatment leads to in-
creased retention of calcium in guinea pig brain.
Numerous studies have evaluated relative effects of lead on mitochonodria of developing
versus adult animals, particularly in the nervous system. Holtzman and Shen Hsu (1976) ex-
posed rat pups at 14 days of age to lead via milk of mothers fed lead in the diet (4 percent
lead carbonate) with exposure lasting for 14 days. A 40 percent increase in state 4 respira-
tory rate of cerebellar mitochonodria was seen within one day of treatment and was lost at the
end of the exposure period. However, at this later time (28 days of age), a substantial inhi-
bition of state 3 respiration was observed. This early effect of lead on uncoupling oxidative
phosphorylation is consistent with the results of Bull et al. (1979) and McCauley et al.
(1979). In these investigations, female rats received lead in drinking water (200 ppm) from
14 days before breeding through weaning of the pups. At 15 days of age, cerebral cortical
slices showed alteration of potassium-stimulated respiratory response and glucose uptake.
Holtzman et al. (1980a) compared mitochondrial respiration in cerebellum and cerebrum in
rat pups exposed to lead beginning at 14 days of age (via milk of mothers fed 4 percent lead
carbonate) and in adult rats maintained on the same diet. Cerebellar mitochondria showed a
very early loss (by 2 days of exposure) of respiratory control in the pups with inhibition of
phosphorylation-coupled respiration for NAD-1inked substrates but not for succinate. Such
changes were less pronounced in mitochondria of the cerebrum and were not seen for either
brain region in the adult rat. This regional-and age-dependency of mitochondrial impairment
parallels features of lead encephalopathy.
In a second study addressing this issue, Holtzman et al. (1981) measured the cytochrome
contents of cerebral and cerebellar mitochondria from rat pups exposed either from birth or at
14 days of age via the same dosing protocol noted above. These were compared to adult animals
exposed in like fashion. Pups exposed to lead from birth showed statistically significant
reductions of cytochrome b, cytochromes c + clf and cytochromes a + a3 in cerebellum by 4
weeks of exposure. Changes in cerebral cytochromes, in contrast, were marginal. When lead
exposure began at 14 days of age, little effect was observed, and adult rats showed little
change. This study indicates that the most vulnerable period for lead effects on developing
brain oxidative metabolism is the same period where a major burst in such activity begins.
-------
Related to effects of lead on energy metabolism in the developing animal mitochondrion is
the effect on brain development. In the study of Bull et al. (1979) noted earlier, cerebral
cytochrome c + cx levels between 10 and 15 days of age decreased in a dose-dependent fashion
at all maternal dosing levels (5-100 mg Pb/liter drinking water) and corresponding blood lead
values for the rat pups (11.7-35.7 ug/dl). Delays in synaptic development in these pups also
occurred, as indexed by synaptic counts taken in the parietal cortex. As the authors con-
cluded, uncoupling of energy metabolism appears to be causally related to delays in cerebral
cortical development.
Consistent with the effects of lead on mitochondrial structure and function are i_n vivo
data demonstrating the selective accumulation of lead in mitochondria. Studies in rats using
radioisotopic tracers 210Pb (Castellino and Aloj, 1969) and 203Pb (Barltrop et al., 1971)
demonstrate that mitochondria will accumulate lead in significant relative amounts, the nature
of the accumulation seeming to vary with the dosing protocol. Sabbioni and Marafante (1976)
as well as Murakami and Hirosawa (1973) also found that lead is selectively lodged in mito-
chondria. Of interest in regard to the effects of lead on brain mitochondria are the data of
Moore et al. (1975a) showing uptake of lead by guinea pig cerebral mitochondria, and the re-
sults of Krigman et al. (1974c) demonstrating that mitochondria in brain from 6-month-old rats
exposed chronically to lead since birth showed the highest uptake of lead (34 percent), fol-
lowed by the nuclear fraction (31 percent). While the possibility of translocation of lead
during subcellular fractionation can be raised, the distribution pattern seen in the reports
of Barltrop et al. (1971) and Castellino and Aloj (1969) over multiple time points makes this
unlikely. Also, findings of jji vivo uptake of lead in brain mitochondria are supported by jn
vitro data discussed below.
12.2.1.4 In Vitro Studies. In vitro studies of both the response of mitochondrial function
to lead and its accumulation by the organelle have been reported, using both isolated mito-
chondria and tissues. Bull (1980) reviewed such data published up to 1979.
Significant reductions in mitochondrial respiration, using both NAD-linked and succinate
substrates, have been reported for isolated heart and brain mitochondria. The lowest levels
of lead associated with such effects appear to be 5 uM or, in some cases, less. Available
evidence suggests that the sensitive site for lead in isolated mitochondria is before cyto-
chrome b in the oxidative chain and involves either tricarboxylic acid enzymes or non-heme
protein/ubiquinone steps. If substrate specificity is compared, e.g., succinate versus glut-
amate/malate oxidation, there appear to be organ-specific differences. As Bull (1980) noted,
tissue-specific effects of lead on cellular energetics may be one basis for differences in
toxicity across organs. Also, several enzymes involved in intermediary metabolism in isolated
mitochondria have been observed to undergo significant inhibition of activity in the presence
of lead; these have been tabulated by Bull (1980).
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One focus of studies dealing with lead effects on isolated mitochondria has been ion
transport—particularly that of calcium. Scott et al. (1971) have shown that lead movement
into rat heart mitochondria involves active transport, with characteristics similar to those
of calcium, thereby establishing a competitive relationship. Similarly, lead uptake into
brain mitochondria is also energy-dependent (Holtzman et al., 1977; Goldstein et al., 1977).
The recent elegant studies of Pounds and coworkers (Pounds et al., 1982a,b), using labeled
calcium or lead and desaturation kinetic studies of these labels in isolated rat hepatocytes,
have elucidated the intracellular relationship of lead to calcium in terms of cellular com-
partmentalization. In the presence of graded amounts of lead (10, 50, or 100 uM), the kinetic
analysis of desaturation curves of 4SCa label showed a lead dose-dependent increase in the
size of all three calcium compartments within the hepatocyte, particularly that deep compart-
ment associated with the mitochondrion (Pounds et al., 1982a). Such changes were seen to be
relatively independent of serum calcium or endogenous regulators of systemic calcium metab-
olism. Similarly, the use of 210Pb label and analogous kinetic analysis (Pounds et al.,
1982b) showed the same three compartments of intracellular distribution as noted for calcium,
including the deep component A: redundant. Hence, there is striking overlap in the cellular
metabolism of calcium and lead. These studies not only further confirm easy entry of lead
into cells and cellular compartments, but also provide a basis for perturbation by lead of
intracellular ion transport, particularly in neural cell mitochondria, where there is a high
capability for calcium transport. Such capability is approximately 20-fold higher than in
heart mitochondria (Nicholls, 1978).
Given the above observations, it is not surprising that a number of investigators have
noted the jji vitro uptake of lead into isolated mitochondria. Walton (1973) noted that lead
is accumulated within isolated rat liver mitochondria over the range of 0.2-100 uM lead;
Walton and Buckley (1977) extended this observation to mitochondria in rat kidney cells in
culture. Electron microprobe analyses of isolated rat synaptosomes (Silbergeld et al., 1977)
and capillaries (Silbergeld et al., 1980b) incubated with lead ion have established that sig-
nificant accumulation of lead, along with calcium, occurs in the mitochondrion. These obser-
vations are consistent with the kinetic studies of Pounds et al. (1982a,b), and the in vitro
data for isolated capillaries are in accord with the observations of Toews et al. (1978), who
found significant lead accumulation in brain capillaries of the suckling rat.
12.2.2 Effects of Lead on the Nucleus
With lead exposure, a cellular reaction typical of many species (including humans) is the
formation of intranuclear lead-containing inclusion bodies, early data for which have been
summarized by Goyer and Moore (1974). In brief, these lead-bearing inclusion bodies A:
(1) have have been verified as to lead content by X-ray microanalysis (Carroll et al., 1970);
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(2) consist of a rather dense core encapsulated by a fibrillary envelope; (3) are a complex
of lead and the acid fractions of nuclear protein; (4) can be disaggregated jin vitro by EDTA;
(5) can appear very rapidly after lead exposure (Choie et al., 1975); (6) consist of a pro-
tein moiety in the complex which is synthesized de novo; and (7) have been postulated to
serve a protective role in the cell, given the relative amounts of lead accumulated and pre-
sumably rendered lexicologically inert.
Based on renal biopsy studies, Cramer et al. (1974) concluded that such inclusion body
formation in renal tubule cells in lead workers is an early response to lead entering the kid-
ney, in view of decreased presence of the inclusion bodies as a function of increased period
of employment. Schumann et al. (1980), however, observed a continued exfoliation of inclu-
sion-bearing tubule cells into urine of workers having a variable employment history.
Any protective role played by the lead inclusion body appears to be an imperfect one, to
the extent that both subcellular organelle injury and lead uptake occur simultaneously with
such inclusion formation, often in association with severe toxicity at the organ system level.
For example, Osheroff et al. (1982) observed lead inclusion bodies in the anterior horn cells
of the cervical spinal cord and neurons of the substantia nigra (as well as in renal tubule
cells) in the adult rhesus monkey, along with damage to the vascular walls and glial processes
and ependymal cell degeneration. At the light- and electron-microscope level, there were no
signs of neuronal damage or altered morphology except for the inclusions. As noted by the
authors, these inclusions in the large neurons of the substantia nigra show that the neuron
will take up and accumulate lead. In the study of Fowler et al. (1980), renal tubule inclu-
sions were observed simultaneously with evidence of structural and functional damage to the
mitochondrion, all at relatively low levels of lead. Hence, it appears that a limited pro-
tective role for these inclusions may extend across a range of lead exposure.
Chromosomal effects and other indices of genotoxicity in humans and animals are discussed
in Section 12.7 of this chapter.
12.2.3 Effects of Lead on Membranes
In theory, the cell membrane is the first organelle to encounter lead, and it is not sur-
prising that cellular effects can be ascribed to interactions at cellular and intracellular
membranes, mainly appearing to be associated with ion transport processes across membranes.
In Section 12.3 a more detailed discussion is accorded the effects of lead on the membrane as
they relate to the erythrocyte in terms of increased cell fragility and increased osmotic re-
sistance. These effects can be rationalized, in large part, by the documented inhibition by
lead of erythrocyte membrane (Na , K )-ATPase.
Lead also appears to interfere with the normal processes of calcium transport across mem-
branes of various tissue types. Silbergeld and Adler (1978) have described lead-induced
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retardation of the release of the neurotransmitter, acetylcholine, in peripheral cholinergic
synaptosomes, due to a blockade of calcium binding to the synaptosomal membrane, reducing
calcium-dependent choline uptake and subsequent release of acetylcholine from the nerve ter-
minal. Calcium efflux from neurons is mediated by the membrane (Na , K )-ATPase via an ex-
change process with sodium. Inhibition of the enzyme by lead, as also occurs with the
erythroctye (see above), increases the concentration of calcium within nerve endings (Goddard
and Robinson, 1976). As seen from the data of Pounds et al. (1982a), lead can also elicit
retention of calcium in neural cells by easy entry into the cell and by directly affecting the
deep calcium compartment within the cell, of which the mitochondrion is a major component.
12.2.4 Other Organellar Effects of Lead
Studies of morphological alterations of renal tubule cells in the rat (Chang et al. ,
1981) and rabbit (Spit et al., 1981) with varying lead treatments have demonstrated lead-
induced lysosomal changes. In the rabbit, with relatively modest levels of lead exposure
(0.25 or 0.5 mg/kg, 3 times weekly over 14 weeks) and corresponding blood lead values of 50
and 60 ug/dl, there was a dose-dependent increase in the amount of lysosomes in proximal con-
voluted tubule cells, as well as increased numbers of lysosomal inclusions. In the rat, expo-
sure to 10 mg/kg i.v. (daily over 4 weeks) resulted in the accumulation of lysosomes, some
gigantic, in the pars recta segment of renal tubules. These animal data are consistent with
observations made in lead workers (Cramer et al., 1974; Wedeen et al., 1975) and appear to
represent a disturbance in normal lysosomal function, with the accumulation of lysosomes being
due to enhanced degradation of proteins arising from effects of lead elsewhere within the
cell.
12.2.5 Summary of Subcellular Effects of Lead
The biological basis of lead toxicity is closely linked to the ability of lead to bind to
ligating groups in biomolecular substances crucial to normal physiological functions. This
binding interferes with physiological processes by such mechanisms as the following: compe-
tition with native essential metals for binding sites, inhibition of enzyme activity, and in-
hibition or other changes in essential ion transport.
The main target organelle for lead toxicity in a variety of cell and tissue types clearly
is the mitochondrion, followed probably by cellular and intracellular membranes. Mitochon-
dria! effects take the form of structural changes and marked disturbances in mitochondrial
function within the cell, especially energy metabolism and ion transport. These effects are
associated, in turn, with demonstrable accumulation of lead in mitochondria, both in vivo and
HI vitro. Structural changes include mitochondrial swelling in many cell types, as well as
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distortion and loss of cristae, which occur at relatively moderate levels of lead exposure.
Similar changes have been documented in lead workers across a wide range of exposure levels.
Uncoupled energy metabolism, inhibited cellular respiration using both succinate and n1-
cotinamide adenine dinucleotide (NAD)-linked substrates, and altered kinetics of intracellular
calcium have been demonstrated i_n vivo using mitochondria of brain and non-neural tissue. In
some cases, relatively moderate lead exposure levels have been associated with such changes,
and several studies have documented the relatively greater sensitivity of this organelle in
young versus adult animals in terms of mitochondrial respiration. The cerebellum appears to
be particularly sensitive, providing a connection between mitochondrial impairment and lead
encephalopathy. Impairment by lead of mitochondrial function in the developing brain has also
been associated with delayed brain development, as indexed by content of various cytochromes.
In the rat pup, ongoing lead exposure from birth is required for this effect to be expressed,
indicating that such exposure must occur before, and is inhibitory to, the burst of oxidative
metabolism activity that normally occurs in the young rat 10-21 days postnatally.
Jji vivo lead exposure of adult rats has also been observed to markedly inhibit cerebral
cortex intracellular calcium turnover (in a cellular compartment that appears to be the mito-
chondrion) at a brain lead level of 0.4 ppm. These results are consistent with a separate
study showing increased retention of calcium in the brains of lead-dosed guinea pigs. A
number of reports have described the jji vivo accumulation of lead in mitochondria of kidney,
liver, spleen, and brain tissue, with one study showing that such uptake was slightly more
than occurred in the nucleus. These data are not only consistent with the various deleterious
effects of lead on mitochondria but are also supported by other, i_n vitro findings.
Significant decreases in mitochondrial respiration jji vitro, using both NAD-linked and
succinate substrates, have been observed for brain and non-neural tissue mitochondria in the
presence of lead at micromolar levels. There appears to be substrate specificity in the inhi-
bition of respiration across different tissues, which may be a factor in differential organ
toxicity. Also, a number of enzymes involved in intermediary metabolism in isolated mitochon-
dria have been observed to undergo significant inhibition of activity with lead.
A major focus of research on lead effects on isolated mitochondria has concerned ion
(especially calcium) transport. Lead movement into brain and other tissue mitochondria, as
does calcium movement, involves active transport. Recent sophisticated kinetic analyses of
desaturation curves for radio!abeled lead or calcium indicate that there is striking overlap
in the cellular metabolism of calcium and lead. These studies not only establish a basis for
easy entry of lead into cells and cell compartments, but also provide a basis for impairment
by lead of intracellular ion transport, particularly in neural cell mitochondria, where the
capacity for calcium transport is 20-fold higher than even in heart mitochondria.
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Lead is also selectively taken up in isolated mitochondria rn vitro, including the mito-
chondria of synaptosomes and brain capillaries. Given the diverse and extensive evidence of
lead's impairment of mitochondrial structure and function as viewed from a subcellular level,
it is not surprising that these derangements are logically held to be the basis of dysfunction
of heme biosynthesis, erythropoiesis, and the central nervous system. Several key enzymes in
the heme biosynthetic pathway are intramitochondrial, particularly ferrochelatase. Hence, it
is to be expected that entry of lead into mitochondria will impair overall heme biosynthesis,
and in fact this appears to be the case in the developing cerebellum. Furthermore, the levels
of lead exposure associated with entry of lead into mitochondria and expression of mitochon-
drial injury can be relatively moderate.
Lead exposure provokes a typical cellular reaction in human and other species that has
been morphologically characterized as a lead-containing nuclear inclusion body. Although it
has been postulated that such inclusions constitute a cellular protection mechanism, such a
mechanism is an imperfect one. Other organelles, e.g., the mitochondrion, also take up lead
and sustain injury in the presence of nuclear inclusion bodies. Chromosomal effects and other
indices of genotoxicity in humans and animals are considered later, in Section 12.7.
In theory, the cell membrane is the first organelle to encounter lead and it is not sur-
prising that cellular effects of lead can be ascribed to interactions at cellular and intra-
cellular membranes in the form of disturbed ion transport. The inhibition of membrane
(Na ,K )-ATPase of erythrocytes as a factor in lead-impaired erythropoiesis is noted else-
where. Lead also appears to interfere with the normal processes of calcium transport across
membranes of different tissues. In peripheral cholinergic synaptosomes, lead is associated
with retarded release of acetylcholine owing to a blockade of calcium binding to the membrane,
while calcium accumulation within nerve endings can be ascribed to inhibition of membrane
(Na+,K+)-ATPase.
Lysosomes accumulate in renal proximal convoluted tubule cells of rats and rabbits given
lead over a wide range of dosing. This also appears to occur in the kidneys of lead workers
and seems to represent a disturbance in normal lysosomal function, with the accumulation of
lysosomes being due to enhanced degradation of proteins because of the effects of lead else-
where within the cell.
Insofar as effects of lead on the activity of various enzymes are concerned, many of the
available studies concern uj vitro behavior of relatively pure enzymes with marginal relevance
to various effects jji vivo. On the other hand, certain enzymes are basic to the effects of
lead at the organ or organ system level, and discussion is best reserved for such effects in
ensuing sections of the document dealing with these systems.
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12.3 EFFECTS OF LEAD ON HEME BIOSYNTHESIS AND ERYTHROPOIESIS/ERYTHROCYTE PHYSIOLOGY IN HUMANS
AND ANIMALS
Lead has well-recognized effects not only on heme biosynthesis, a crucial process common
to many organ systems, but also on the formation and physiology of erythrocytes. This section
is therefore divided for purposes of discussion into the following: (1) effects of lead on
heme biosynthesis including discussion of interrelationships between heme biosynthesis im-
pairment and (a) interference with vitamin-D metabolism and (b) certain neurotoxic effects of
lead; and (2) effects of lead on erythropoiesis and erythrocyte physiology. Discussion of the
latter is further subdivided into effects of lead on hemoglobin production, cell morphology
and survival, and erythropoietic nucleotide metabolism.
12.3.1 Effects of Lead on Heme Biosynthesis
The effects of lead on heme biosynthesis are very well known because of their prominence
and the large number of studies of these effects in humans and experimental animals. In addi-
tion to being a constituent of hemoglobin, heme is a prosthetic group of a number of tissue
hemoproteins having diverse functions, such as myoglobin, the P-450 component of the mixed-
function oxidase system, and the cytochromes of cellular energetics. Hence, any effects of
lead on heme biosynthesis will, perforce, pose the potential for multi-organ toxicity.
At present, much of the available information concerning the effects of lead on heme bio-
synthesis has been obtained by measurements in blood, due in large part to the relative ease
of access to blood and in part to the fact that blood is the vehicle for movement of metabo-
lites from other organ systems. On the other hand, a number of reports have been concerned
with lead effects on heme biosynthesis in tissues such as kidney, liver, and brain. In the
discussion below, various steps in the heme biosynthetic pathway affected by lead are discus-
sed separately, with information describing erythropoietic effects usually appearing first,
followed by studies involving other tissues.
The process of heme biosynthesis results in formation of a porphyrin, protoporphyrin IX,
starting with glycine and succinyl-coenzyme A. Heme biosynthesis culminates with the inser-
tion of iron at the center of the porphyrin ring. As may be noted in Figure 12-1, lead inter-
feres with heme biosynthesis by disturbing the activity of three major enzymes: (1) it in-
directly stimulates, by feedback derepression, the mitochondria! enzyme delta-aminolevulinic
acid synthetase (ALA-S), which mediates the condensation of glycine and succinyl-coenzyme A to
form delta-aminolevulinic acid (ALA); (2) it directly inhibits the cytosolic enzyme delta-
aminolevulinic acid dehydrase (ALA-D), which catalyzes the cyclocondensation of two units of
ALA to porphobilinogen; (3) it disturbs the mitochondrial enzyme ferrochelatase, found in
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MITOCHONDRION
MITOCHONDRIA!. MEMBRANE
GLYCINE
-f
SUCCINYL-CoA
HEME
ALA SYNTHETASE
(INCREASE)
FERRO-
CHELATASE
IRON+PROTOPORPHYRIN
I
Pb (DIRECTLY OR 1
BY DEREPRESSION) *
AMINOLEVULINIC ACID
(ALA)
IRON
ALA
DEHYDRASE
(DECREASE)
Pb
•Pb
COPROPORPHYRIN
(INCREASE)
PORPHOBILINOGEN
Figure 12-1. Effects of lead (Pb) on heme biosynthesis.
liver, bone marrow, and other tissues, either by direct inhibition or by alteration of intra-
mitochondrial transport of iron. Ferrochelatase catalyzes the insertion of iron (II) into the
protoporphyrin ring to form heme and is situated in mammals in the inner mitochondrial mem-
brane (McKay et al., 1969).
12.3.1.1 Effects of Lead on Delta-Aminolevulim'c Acid Synthetase. The activity of the enzyme
ALA-S is the rate-limiting step in the heme biosynthetic pathway. With decreased heme forma-
tion at other steps downstream or with increased heme oxygenase activity, a compensatory in-
crease of ALA-S activity occurs through feedback derepression and enhances the rate of heme
formation. Hence, excess ALA formation is due to both stimulation of ALA-S and direct inhibi-
tion of ALA-D (see below).
Increased ALA-S activity has been reported in lead workers (Takaku et al., 1973; Campbell
et al., 1977; Meredith et al. , 1978), with leukocyte ALA-S reported to be stimulated at a
blood lead value of 40 ug/dl (Meredith et al., 1978), a level at which ALA-D activity is sig-
nificantly inhibited. To the extent that mitochondria in leukocytes show a dose-effect rela-
tionship comparable to the bone marrow and hepatic systems, it appears that most of the excess
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ALA formation below the observed threshold value is due to ALA-D inhibition. From the
authors' data, blood ALA had increased about twofold in a subset of the worker population over
the blood lead range of 18-40 ug/dl.
In vitro and j_n vivo experimental data have provided organ-specific results in terms of
the direction of the effect of lead on ALA-S activity. Silbergeld et al. (1982) observed that
ALA-S activity was increased in kidney with acute lead exposure in rats, while chronic treat-
ment was associated with increased activity of the enzyme in spleen. In liver, however, ALA-S
activity was reduced under both acute and chronic dosing. Fowler et al. (1980) reported that
renal ALA-S activity was significantly reduced in rats continuously exposed to lead _m utero,
through development, and up to 9 months of age. Meredith and Moore (1979) noted a steady in-
crease in hepatic ALA-S activity when rats were given lead parenterally over an extended
period of time. Maxwell and Meyer (1976) and Goldberg et al. (1978) also noted increased
ALA-S activity in rats given lead parenterally. It appears that the type and timeframe of
dosing influence the observed effect of lead on the enzyme activity. Using a rat liver cell
line (RLC-GAI) in culture, Kusell et al. (1978) demonstrated that lead could produce a time-
dependent increase in ALA-S activity. Stimulation of activity was observed at lead levels as
low as 5 x 10 M, with maximal stimulation at 1 x 10* M. The authors reported that the
increase in activity was associated with the biosynthesis of more enzyme rather than with
stimulation of the pre-existing enzyme. Lead-stimulated ALA-S formation was also not limited
to liver cells; rat gliomas and mouse neuroblastomas showed similar results.
12.3.1.2 Effects of Lead on Delta-Aminolevulinic Acid Dehydrase and Delta-Aminolevulinic Acid
Accumulati on/Excreti on. Delta-aminolevulinic acid dehydrase (5-aminolevulinate hydrolase;
porphobilinogen synthetase; E.G. 4.2.1.24; ALA-D) is a sulfhydryl, zinc-requiring allosteric
enzyme in the heme biosynthetic pathway that catalyzes the conversion of two units of ALA to
porphobilinogen. The enzyme's activity is very sensitive to inhibition by lead, but the inhi-
bition is reversed by reactivation of the sulfhydryl group with agents such as dithiothreitol
(Granick et al., 1973), zinc (Finelli et al., 1975), or zinc plus glutathione (Mitchell et
al., 1977).
The activity of ALA-D appears to be inhibited at virtually all blood lead levels studied
so far, and any threshold for this effect remains to be identified (see discussion below).
Dresner et al. (1982) found that ALA-D activity in rat bone marrow suspensions was signifi-
cantly inhibited to 35 percent of control levels in the presence of 5 x 10 M (0.5 |jM) lead.
This potency was unmatched on a comparative molar basis by any other metal tested. Recently,
Fujita et al. (1981) showed evidence of an increase in the amount of ALA-D in erythrocytes in
lead-exposed rats that was ascribed to an increased rate of ALA-D synthesis in bone marrow
cells. Hence, the commonly observed net inhibition of activity occurs even in the face of an
increase in ALA-D synthesis.
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Hernberg and Nikkanen (1970) found that enzyme activity was correlated inversely with
blood lead values in a group of urban, nonexposed subjects. Enzyme activity was inhibited 50
percent at a blood lead level of 16 ug/dl. Other reports have confirmed these observations
across age groups and exposure categories (Alessio et al., 1976b; Roels et al., 1975b; Nieburg
et al., 1974; Wada et al., 1973). A ratio of activated to inhibited enzyme activity (versus a
single activity measurement, which does not accommodate intersubject genetic variability) mea-
sured against children's blood lead values of 20-90 ug/dl was employed by Granick et al.
(1973) to obtain an estimated threshold of 15 ug/dl for an effect of lead. On the other hand,
Hernberg and Nikkanen (1970) observed no threshold in their subjects, all of whom were at or
below 16 ug/dl. Note that the lowest blood lead actually measured by Granick et al. (1973)
was higher than the values measured by Hernberg and Nikkanen (1970).
Kuhnert et al. (1977) reported that ALA-D activity measures in erythrocytes from both
pregnant women and cord blood of infants at delivery are inversely correlated with the cor-
responding blood lead values, using the activated/inhibited activity ratio method of Granick
et al. (1973). The correlation coefficient of activity with lead level was higher in fetal
erythrocytes (r = -0.58, p <0.01) than in the mothers (r = -0.43, p <0.01). The mean inhi-
bition level was 28 percent in mothers versus 12 percent in the newborn. A study by Lauwerys
et al. (1978) in 100 pairs of pregnant women and infant cord blood samples confirms this
observation, i.e., for fetal blood r = 0.67 (p <0.001) and for maternal blood r = -0.56
(p <0.001).
While several factors other than lead may affect the activity of erythrocyte ALA-D, much
of the available information suggests that most of these factors do not materially compromise
the interpretation of the relationship between enzyme activity and lead or the use of this
relationship for screening purposes. Border et al. (1976) questioned the reliability of ALA-D
activity measurement in subjects concurrently exposed to lead and zinc because zinc also
affects the activity of the enzyme. The data of Meredith and Moore (1980) refute this objec-
tion. In unexposed subjects who had serum zinc values of 80-120 uM, there was only a minor
activating effect with increasing zinc when contrasted to the correlation of activity and
blood lead in these same subjects. In workers exposed to both lead and zinc, serum zinc
values were greater than in subjects with just lead exposure, but the mean level of enzyme ac-
tivity was still much lower than in controls (p <0.001).
The preceding discussion indicates that neither variability within the normal range of
physiological zinc in humans nor combined excessive zinc and lead exposure in workers materi-
ally affects ALA-D activity. The obverse of this, lead exposure in the presence of zinc defi-
ciency, is probably a more significant issue, but one that has not been well studied. Since
ALA-D is a zinc-requiring enzyme, one would expect that optimal activity would be governed by
j_n vivo zinc availability. Thus, zinc deficiency could potentially have a dual deleterious
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effect on ALA-D activity: first, a direct reduction in ALA-D activity through reduced zinc
availability, and second, an indirect and further inhibition of ALA-D activity because of
enhanced lead absorption in the presence of zinc deficiency (see Chapter 10, Section 10.5).
The recent study of Roth and Kirchgessner (1981) indicates that ALA-D activity is signi-
ficantly decreased in the presence of zinc deficiency. In zinc-deficient rats showing reduced
serum and urinary zinc levels, the level of erythrocyte ALA-D activity was only 50 percent
that of pair-fed controls, while urinary ALA was significantly elevated. Although these in-
vestigators did not measure blood lead in deficient and control animal groups, it would appear
that the level of inhibition is more than can be accounted for just on the basis of increased
lead absorption from the diet. Given the available information documenting zinc deficiency in
children (Section 10.5) as well as the animal study of Roth and Kirchgessner (1981), the rela-
tionship of lead, zinc deficiency, and ALA-D activity in young children merits further, care-
ful study.
Moore and Meredith (1979) noted the effects of carbon monoxide on the activity of ALA-D,
comparing moderate or heavy smokers with nonsmokers. At the highest level of carboxyhemoglo-
bin measured in their smoker groups, the depression of ALA-D activity was 2.1 percent. In
these subjects, a significant inverse correlation of ALA-D activity and blood lead existed,
but there was no significant correlation of such activity and blood carboxyhemoglobin levels.
While blood ethanol has been reported to affect ALA-D activity (Moore et al., 1971;
Abdulla et al., 1976), its effect is significant only under conditions of acute alcohol intox-
ication. Hence, relevance of this observation to screening is limited, particularly in
children. The effect is reversible, declining with clearing of alcohol from the blood stream.
Lead-induced inhibition of ALA-D activity in erythrocytes apparently reflects a similar
effect in other tissues. Secchi et al. (1974) observed a clear correlation in 26 lead workers
between hepatic and erythrocyte ALA-D activity as well as the expected inverse correlation
between such activity and blood lead in the range of 12-56 ug/dl. In suckling rats, Millar
et al. (1970) noted decreased enzyme activity in brain and liver as well as erythrocytes when
lead was administered orally. In the study of Roels et al. (1977), tissue ALA-D changes were
not observed when dams were administered 1, 10, or 100 ppm lead in drinking water. However,
the data of Roels et al. (1977) may reflect a lower effective dose taken in by the dams and
delivered to the rat pups in maternal milk, because the pups showed no tissue enzyme activity
changes. Silbergeld et al. (1982) described moderate inhibition of ALA-D activity in brain
and significant inhibition in kidney, liver, and spleen when adult rats were acutely exposed
to lead given intraperitoneally; chronic exposure was associated with reduced activity in
kidney, liver, and spleen. Gerber et al. (1978) found that neonatal mice exposed to lead from
birth through 17 days of age at a level of 1.0 mg/ml in water showed significant decreases in
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brain ALA-D activity (p <0.01) at all time points studied. These results support the data of
Millar et al. (1970) for the suckling rat. In the study by Millar et al., rats exposed from
birth through adulthood only showed significant decreases of brain ALA-D activity at 15 and 30
days; this finding also supports other data for the developing rodent. It would appear,
therefore, that brain ALA-D activity is more sensitive to lead in the developing animal than
in the adult.
The study of Dieter and Finley (1979) sheds light on the relative sensitivity of ALA-D
activity in several regions of the brain and permits comparison of blood versus brain ALA-D
activity as a function of lead level. Mallard ducks given a single pellet of lead showed 1
ppm lead in blood, 2.5 ppm lead in liver, and 0.5 ppm lead in brain by 4 weeks. Cerebellar
ALA-D activity was reduced by 50 percent at a lead level below 0.5 ppm; erythrocyte enzyme
activity was lowered by 75 percent. Hepatic ALA-D activity was comparable to cerebellar acti-
vity or somewhat less, although the lead level in the liver was fivefold higher. Cerebellar
ALA-D activity was significantly below that for cerebrum. In an avian species, then, at blood
lead levels at which erythrocyte ALA-D activity was significantly depressed, activity of the
enzyme in cerebellum was even more affected relative to lead concentration.
The inhibition of ALA-D is reflected by increased levels of its substrate, ALA, in urine
(Haeger, 1957) as well as in whole blood or plasma (O'Flaherty et al., 1980; Meredith et al.,
1978; MacGee et al. , 1977; Chisolm, 1968; Haeger-Aronsen, 1960). Cramer et al. (1974) demon-
strated that ALA clearance into urine parallels glomerular filtration rate across a range of
lead exposures, suggesting that increased urinary output with increasing circulating ALA is
associated with decreased tubular reabsorption (Moore et al., 1980). Based on their measure-
ments of plasma and urinary ALA across a range of blood lead in adults, O'Flaherty et al.
(1980) calculated a mean fractional reabsorption of 40 percent for ALA. Tubular secretion
also occurs. Reabsorption appears to be saturable. In rats, fractional reabsorption was much
higher, 90-99 percent.
The detailed study of Meredith et al. (1978), which involved both control subjects and
lead workers, indicated that in elevated lead exposure the increase in urinary ALA is preceded
by a significant rise in circulating levels of ALA. The overall relationship of plasma ALA to
blood lead was exponential and showed a perceptible continuity of the correlation even down to
the lowest blood lead value of the control group, 18 pg/dl. The relationship of plasma ALA to
urinary levels of the precursor was found to be exponential, indicating that as plasma ALA
increases, a greater proportion of ALA undergoes excretion into urine. Inspection of the plot
of urinary versus plasma ALA in these subjects shows that the correlation persists down to the
plasma ALA concentration corresponding to the lowest blood lead level, 18 ug/dl. These
results are contradicted by those of O'Flaherty et al. (1980), who showed no correlation of
blood lead with plasma ALA below a value of 40 ug/dl. A key factor in these contradictory
12-18
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studies is the method of ALA measurement. Meredith et al. derived their data from a colori-
metric technique that measures ALA as well as other aminoketones, while such aminoketones are
not detected in the gas-liquid chromatography method used by 0'Flaherty et al. Although the
measurements of 0'Flaherty et al. are generally more specific for ALA in plasma than any
colorimetric technique, their validity at low plasma ALA levels remains to be established in
the field (see Chapter 9). The blood (plasma) ALA values reported by Meredith et al. were
generally higher than those measured by 0'Flaherty et al. and appeared to be high in terms of
ALA renal clearance rates. ALA is, however the only aminoketone studied so far that corre-
lates with lead directly. Aminoacetone, also measured in the Meredith et al. study, is a
metabolite of an ami no acid and is not known to be affected by lead exposure. Thus, notwith-
standing a positive bias in absolute ALA values, the relative changes in ALA would appear to
provide the most plausible basis for the observed correlation with blood lead levels as low as
18 ug/dl in the study by Meredith et al.
Urinary ALA has been employed extensively as an indicator of excessive lead exposure,
particularly in occupational settings (e.g., Davis et al., 1968; Selander and Cramer, 1970;
Alessio et al., 1976a). The reliability of this test in initial screening of children for
lead exposure has been questioned by Specter et al. (1971) and Blanksma et al. (1969), who
pointed out the failure of urinary ALA analysis to detect lead exposure when compared with
blood lead values. This is due to the fact that an individual subject will show a wide vari-
ation in urinary ALA with random sampling. Chisolm et al. (1976) showed that reliable levels
could only be obtained with 24-hr collections. In children with blood lead levels above
40 ug/dl, the relationship of ALA in urine to blood lead becomes similar to that observed in
lead workers (see below).
A correlation exists between blood lead and the logarithm of urinary ALA in workers
(Meredith et al., 1978; Alessio et al., 1976a; Roels et al., 1975a; Wada et al., 1973;
Selander and Cramer, 1970) and in children (National Academy of Sciences, 1972). Selander and
Cramer (1970) reported that two different correlation curves were obtained, one for individ-
uals below 40 ug/dl blood lead and a different one for values above this, although the degree
of correlation was less than with the entire group. A similar observation has been reported
by Lauwerys et al. (1974) from a study of 167 workers with blood lead levels of 10-75 ug/dl.
Meredith et al. (1978) found that the correlation curve for blood ALA versus urinary ALA
was linear below a blood lead of 40 Mfl/dl, as was the relationship of blood ALA to blood lead.
Hence, there was also a linear relationship between blood lead and urinary ALA below 40 ug/dl,
i.e., a continuation of the correlation below the commonly accepted threshold blood lead value
of 40 ug/dl (see below). Tsuchiya et al. (1978) have questioned the relevance of using single
correlation curves to describe the blood lead-urinary ALA relationship across a broad range of
12-19
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exposure, because they found that this relationship in workers showing moderate, intermediate,
and high lead exposure could be described by three correlation curves with different slopes.
This finding is consistent with the observations of Selander and Cramer (1970) as well as the
results of Meredith et al. (1978) and Lauwerys et al. (1974). Chisolm et al. (1976) described
an exponential correlation between blood lead and urinary ALA in children 5 years old or
younger, with blood lead levels ranging from 25 to 75 (jg/dl. The upward slope in the regres-
sion line appears to be most pronounced at a blood lead level of about 40 ug/dl, but the
correlation may persist below this level. In adolescents with blood lead below 40 ug/dl, no
clear correlation was observed.
It is apparent from the above reports (Tsuchiya et al. , 1978; Meredith et al., 1978;
Selander and Cramer, 1970) that circulating ALA and urinary ALA levels are elevated and corre-
lated at blood lead values below 40 ug/dl in humans. These findings are consistent, as shown
in the Meredith et al. (1978) study, with the significant and steady increase in ALA-0 inhibi-
tion concomitant with rising blood levels of ALA, even at blood lead values considerably below
40 ug/dl. Increases of ALA in tissues of experimental animals exposed to lead have also been
documented. In the study of Silbergeld et al. (1982), acute administration of lead at a
rather high dose to adult rats was associated with an elevation in spleen and kidney ALA com-
pared to that of controls, while in chronic exposure there was a moderate increase in ALA in
the brain and a large increase (9-fold to 15-fold) in kidney and spleen. Liver levels with
either form of exposure were not materially affected, although there was inhibition of liver
ALA-D, particularly in the acute dose group.
12.3.1.3 Effects of Lead on Heme Formation from Protoporphyrin. The accumulation of proto-
porphyrin in the erythrocytes of individuals with lead intoxication has been recognized since
the 1930s (Van den Bergh and Grotepass, 1933), but it has only recently been possible to study
this effect through the development of sensitive and specific analytical techniques that per-
mit quantitative measurement. In particular, the development of laboratory microtechniques
and the hematofluorometer has allowed the determination of dose-effect relationships as well
as the use of such measurements to screen for lead exposure.
In humans under normal circumstances, about 95 percent of the protoporphyrin in cir-
culating erythrocytes is zinc protoporphyrin (ZPP), with the remaining 5 percent present as
"free" protoporphyrin (Chisolm and Brown, 1979). Accumulation of protoporphyrin IX in the
erythrocytes is the result of impaired iron (II) placement in the porphyrin moiety to form
heme, an intramitochondrial process. In lead exposure, the porphyrin acquires a zinc ion in
lieu of the native iron; the resulting ZPP is tightly bound in the available heme pockets for
the life of the erythrocyte, about 120 days (Lamola et al., 1975a,b). Hammond and coworkers
(1985) recently observed that in a group of young children aged 3-36 months (n = 165) the
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fraction of ZPP versus total erythrocyte protoporphyrin (EP) varied with age; it was at a min-
imum at 3 months and approached unity at 33 months. The basis for the age-related instability
of this ratio may be either a biological factor or an artifact of analytical methodology. A
plausible biological basis is that zinc bioavailability and zinc nutritional status (subopti-
mal in the early age groups) determine the extent of zinc placement in EP. The significance
of these observations for EP screening of very young children has been noted in Chapter 9.
In lead poisoning, the accumulation of protoporphyrin differs from that seen in the gene-
tically transmitted disorder erythropoietic protoporphyria. In the latter case, there is a
defect in ferrochelatase function, i.e., enzyme function is only 10-25 percent of normal
(Bloomer, 1980), leading to loose attachment of the porphyrin, accumulated without uptake of
zinc, on the surface of the hemoglobin. Loose attachment permits diffusion into plasma and
ultimately into the skin, where photosensitivity is induced. This behavior is absent in
lead-associated porphyrin accumulation. The two forms of porphyrin, free and zinc-containing,
differ sufficiently in fluorescence spectra to permit a laboratory distinction. With iron
deficiency, there is also accumulation of protoporphyrin as the zinc complex in the heme
pocket; this resembles in large measure the characteristics of lead intoxication.
The elevation of erythrocyte ZPP has been extensively documented as exponentially corre-
lated with blood lead in children (Piomelli et al., 1973; Kammholz et al., 1972; Sassa et al.,
1973; Lamola et al., 1975a,b; Roels et al., 1976) and in adult workers (Valentine et al.,
1982; Lilis et al., 1978; Grandjean and Lintrup, 1978; Alessio et al., 1976b; Roels et al.,
1975a, 1979; Lamola et al., 1975a,b). Reigart and Graber (1976) and Levi et al. (1976) have
demonstrated that ZPP elevation can predict which children tend to increase their blood lead
levels, a circumstance that probably rests on the nature of chronic lead exposure in certain
groups of young children where a pulsatile blood lead curve is superimposed on some level of
ongoing intake of lead that continues to elevate the ZPP values.
Accumulation of ZPP only occurs in erythrocytes formed during lead's presence in erythro-
poietic tissue. This results in a lag of several weeks before the fraction of new ZPP-rich
cells is large enough to influence total cell ZPP level. On the other hand, elevated ZPP in
erythrocytes long after significant lead exposure has ceased appears to be a better indicator
of resorption of stored lead in bone than other measurements. Alessio et al. (1976b) reported
that former lead workers removed from exposure at the workplace for more than 12 months in all
cases still showed the typical logarithmic correlation between ZPP and blood or urinary lead.
However, the best correlation was observed between ZPP and chelatable lead, that fraction of
the total body burden considered toxicologically active (see Chapter 10). This post-exposure
relationship for adults clearly indicates that significant levels of hematologically toxic
lead continue to circulate long after exposure to lead has ceased.
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In a report relevant to the problem of multiple-indicator measurements in the assessment
of the degree of lead exposure, Hesley and Wimbish (1981) studied changes in blood lead and
ZPP in two groups: newly exposed lead workers and those removed from significant exposure.
In new workers, blood lead achieved a plateau at 9-10 weeks, while ZPP continued to rise over
the entire study interval of 24 weeks. Among workers removed from exposure, both blood lead
and ZPP values remained elevated up to the end of this study period (33 weeks), but the
decline in ZPP concentration lagged behind blood lead in reaching a plateau. These investiga-
tors logically concluded that the difficulty in demonstrating reliable blood lead-ZPP rela-
tionships may reflect differences in the time at which the two measures reach plateau. The
authors also suggested that more reliance should therefore be placed on ZPP than on blood lead
levels before permitting re-entry into areas of elevated lead exposure.
The threshold for the effect of lead on ZPP accumulation is affected by the relative
spread of blood lead values and the corresponding concentrations of ZPP. In many cases these
range from "normal" levels in nonexposed subjects to values reflecting considerable exposure.
Furthermore, iron deficiency is also associated with ZPP elevation, particularly in children
2-3 years old or younger.
For EP elevation in adults, Roels et al. (1975b) found that the relationship of this
effect to blood lead ended at 25-30 ug/dl, confirmed by the log-transformed data of Joselow
and Flores (1977), Grandjean and Lintrup (1978), Odone et al. (1979), and Herber (1980). In
children 10-15 years of age, the data of Roels et al. (1976) indicate an effect threshold of
15.5 ug/dl. In this study the threshold was taken as the point of intersection of two re-
gression lines derived from two groups of children. The population dose-response relationship
between EP and blood lead in these children indicated that EP levels were significantly higher
(>2 standard deviations) than the reference mean in 50 percent of the children at a blood lead
level of 25 ug/dl. In the age range of children studied here, iron deficiency is uncommon and
these investigators did not note any significant hematocrit change in the exposure group. In
fact, hematocrit was lower in the control group, although these subjects had lower ZPP levels.
In this study, then, iron deficiency was unlikely to have been a confounding factor in the
primary relationship. Piomelli et al. (1977) obtained a comparable threshold value (15.5
ug/dl) for lead's effect on ZPP elevation in children who were older than 4 years as well as
those who were 2-4 years old. If iron deficiency was a factor in the results for this large
study population (1816 children), one would expect a greater impact in the younger group,
where the deficiency is more common.
Within the blood lead range considered "normal," i.e., below 30-40 (jg/dl, assessment of
any ZPP-blood lead relationship is strongly influenced by the relative analytical proficiency
of the laboratory carrying out both measurements, particularly for blood lead at lower values.
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The type of statistical treatment of the data is also a factor, as are some biological sources
of variability. With respect to subject variability, Grandjean (1979) has documented that ZPP
increases throughout adulthood, while hemoglobin remains relatively constant. Hence, age
matching is a prerequisite. Similarly, the relative degree of ZPP response depends on gender:
females show a greater response for a given blood lead level than do males (see discussion
below).
Suga et al. (1981) claimed no apparent correlation between blood lead levels below
40 ug/dl and blood ZPP content in an adult population of 395 male and female subjects. The
values for males and females were combined because of no measured differences in ZPP response,
which is at odds with the studies of Stuik (1974), Roels et al. (1975b), Zielhuis et al.
(1978a,b), Odone et al. (1979), and Toriumi and Kawai (1981). Also, EP was found to increase
with increasing age, despite the fact that the finding of no correlation between blood lead
and ZPP was based on a study population with all age groups combined.
Piomelli et al. (1982) investigated both the threshold for the effect of lead on EP ac-
cumulation and a dose-response relationship in 2004 children, 1852 of whom had blood lead
values below 30 pg/dl. In this study, blood lead and EP measurements were done in facilities
with a high proficiency for both blood lead and ZPP analyses. The study employed two statis-
tical approaches (segmental line techniques and probit analysis), both of which revealed an
average threshold blood lead level of 16.5 ug/dl in the full group and in the children with
blood lead values below 30 ug/dl. In this report, the effect of iron deficiency and other
non-lead factors was tested and removed using the Abbott formula (Abbott, 1925). With respect
to population dose-response relationships, it was found that blood lead values of 28.6 and
SS.^MQ/d1 corresponded to significant EP elevation of more than 1 or 2 standard deviations,
respectively, above a reference mean in 50 percent of the subjects. At a blood lead level of
30 ug/dl, furthermore, it was determined that 27 percent of children would have an EP greater
than 53 ug/dl.
In a related study (Rabinowitz et al., 1986), simultaneous blood lead, ZPP, and hemato-
crit measurements were made semi annually on 232 normal infants during their first two years of
life. The incidence of elevated ZPP (mean + 1 or 2 S.D.) was unrelated to blood lead below 15
Mg/dl but was 4-fold greater above this threshold. The relationship persisted after correc-
tion for the small number (4 percent) of infants with a hematocrit below 33 percent. This
survey extends the observations of Piomelli et al. (1982) to a younger and less lead-burdened
population.
Comparison of EP elevations among adult males and females and children at a given blood
lead level generally indicates that children and adult females are more sensitive to this ef-
fect of lead. Lamola et al. (1975a,b) demonstrated that the slope of ZPP versus blood lead
12-23
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was steeper in children than in adults. Reels et al. (1976) found that women and children
were equally more sensitive in response than adult males, a finding also observed in the popu-
lation studied by Odone et al. (1979). Other comparisons between adults, either as groups
studied at random or in a voluntary lead exposure study, also document the sensitivity of
females over males to this effect of lead (Stuik, 1974; Roels et al., 1975b, 1976, 1979;
Toriumi and Kawai, 1981). The heightened response of females to lead-associated EP elevation
has also been investigated in rats (Roels et al., 1978a) and has been shown to be related to
hormonal interactions with lead, thus confirming the human data of Roels et al. (1975b, 1976,
1979) that iron status is not a factor in the phenomenon.
The effect of lead on iron incorporation into protoporphyrin in the heme biosynthetic
pathway is not restricted to the erythropoietic system. Evidence of a generalized effect of
lead on tissue heme synthesis at low levels of lead exposure comes from the recent studies of
Rosen and coworkers (Rosen et al., 1980, 1981; Mahaffey et al., 1982) concerning lead-asso-
ciated reductions in 1,25-dihydroxyvitamin D (1,25-(OH)2D) (see Section 12.3.5). Such re-
ductions probably occur because lead has an inhibitory effect on renal 1-hydroxylase, a heme-
requiring cytochrome P-450 mediated mitochondrial enzyme system that converts 25-hydroxy-
vitamin D to 1,25-(OH)2D. In an independent study, it has been shown in animals chronically
exposed to moderate amounts of lead that kidney ferrochelatase activity is inhibited with
elevation of EP, reducing the kidney heme pool for heme-requiring enzymes (Fowler etal.,
1980). The low end of the blood lead range associated with lowered 1,25-(OH)2D levels and
inhibited 1-hydroxylase activity corresponds to the level of lead associated with the onset of
EP accumulation in erythropoietic tissue (see above). Sensitivity of renal mitochondrial
1-hydroxylase activity to lead is consistent with a large body of information showing the
susceptibility of renal tubule cell mitochondria to injury by lead and with the chronic lead
exposure animal model of Fowler et al. (1980), discussed in more detail below.
Formation of the heme-containing protein cytochrome P-450, which is an integral part of
the hepatic mixed-function oxygenase system, has been documented in animals (Alvares et al.,
1972; Scoppa et al., 1973; Chow and Cornish, 1978; Goldberg et al., 1978; Meredith and Moore,
1979) and humans (Alvares et al., 1975; Meredith et al. , 1977; Fischbein et al., 1977; Saenger
et al., 1984) as being affected by lead exposure, particularly acute lead intoxication. Many
of these studies used altered drug detoxification rates as a functional measure of such
effects. In the work of Goldberg et al. (1978), increasing the level of lead exposure in rats
was correlated with both a steadily decreasing P-450 content of hepatic microsomes and decre-
ased activity of the detoxifying enzymes aniline hydroxylase and aminopyrine demethylase.
Similarly, the data of Meredith and Moore (1979) showed that continued dosing of rats with
lead results in steadily decreased microsomal P-450 content, decreased total heme content of
microsomes, and increased ALA-S activity.
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Recently, Saenger and coworkers (1984) demonstrated that there was significantly reduced
6B-hydroxylation of cortisol in children having a positive ethylenediaminetetraacetic acid
(EDTA) provocation test compared to a negative test group, under conditions of age matching
and controlling for free cortisol. Because 6B-hydroxycortisol formation is mediated by
hepatic cytochrome P-450 microsomal monooxygenase, lead appears to inhibit this system at
relatively moderate levels of lead exposure in children.
According to Litman and Correia (1983), treatment of rats with either the organic
porphyrinic agent 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-l,4-dihydropyridine (DDEP) or in-
organic lead is associated with an inhibition of the hepatic enzyme system tryptophan
pyrrolase, owing to depletion of the hepatic heme pool and resulting in elevated levels of
tryptophan, serotonin, and 5-hydroxyindoleacetic acid in the brain. With infusion of heme,
however, brain levels were restored to normal. These studies were carried out with
phenobarbital induction of the enzyme system. The behavior of lead alone was not
investigated.
Of interest in this regard are data relating to neural tissue. Studies of organotypic
chick dorsal root ganglion in culture document that the nervous system has heme biosynthetic
capability (Whetsell et al., 1978) and that this cell system elaborates decreased amounts of
porphyrinic material in the presence of lead (Sassa et al., 1979). In a later investigation,
Whetsell and coworkers (Whetsell and Kappas, 1981; Whetsell et al., 1984) reported that mouse
dorsal root ganglion in culture exposed to lead for 6 weeks at 10" M (2 ug Pb/ml medium)
showed progressive severe destruction of myelin and Schwann cells as well as alterations in
axonal and neuronal ultrastructure. Because the co-administration of heme (10 M) prevented
most of these destructive effects, particularly in Schwann cells, axons, and neurons, there is
an indication of a relationship between inhibition by lead of heme biosynthesis in neural tis-
sue and the morphological changes observed.
Chronic administration of lead to neonatal rats indicates that at low levels of exposure,
with modest elevations of blood lead, there is a retarded growth in the respiratory chain
hemoprotein cytochrome C and disturbed electron transport function in the developing rat cere-
bral cortex (Bull et al. , 1979). The study of Holtzman et al. (1981) indicates that the cyto-
chrome group affected and the brain region affected appear to differ with the age of the young
animal at the start of dosing and the duration of dosing. All measured changes involved
reduction at the p <0.05 level. Young rats fed lead from birth for 3 weeks showed reduction
in cytochrome aa3 of cerebral mitochondria, while feeding for 4 weeks showed reduction in all
cerebellar mitochondrial cytochromes. When feeding commenced at 2 weeks, the range of effects
also depended on duration of exposure, with reduction of cytochrome b in cerebral mitochondria
after one week, reduction in cytochrome c + ^ in cerebral mitochondria after 2 weeks, and
cerebellar cytochrome c + Cj reduction after two weeks. These effects on the developing
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organism are accentuated by increased whole body lead retention in both developing children
and experimental animals as well as by higher retention of lead in the brain of suckling rats
as compared to adults.
Heme oxygenase activity is elevated in lead-intoxicated animals (Maines and Kappas, 1976;
Meredith and Moore, 1979) in which relatively high dosing is employed. This indicates that
normal repression of the enzyme's activity is lost, further adding to heme reduction and loss
of regulatory control on the heme biosynthetic pathway.
The mechanism(s) underlying derangement of heme biosynthesis leading to ZPP accumulation
in lead intoxication can be ascribed to impaired mitochondrial transport of iron, ferrochela-
tase inhibition, or a combination of both. Lead-induced effects on mitochondrial morphology
and function, which are well known (Goyer and Rhyne, 1973; Fowler, 1978), may include impaired
iron transport (Borova et al., 1973). Moreover, the resemblance of lead-associated ZPP accu-
mulation to a similar effect of iron deficiency is consistent with the unavailability of iron
to ferrochelatase rather than with direct enzyme inhibition. However, the porphyrin pattern
seen in the congenital disorder erythropoietic porphyria, where ferrochelatase itself is af-
fected, is different from that seen in lead intoxication.
Several animal studies indicate that the effects of lead on heme formation may involve
both ferrochelatase inhibition and impaired mitochondrial transport of iron. Hart et al.
(1980) observed that acute lead exposure in rabbits is associated with a two-stage hema-
topoietic response: an earlier phase that results in significant formation of free versus
zinc protoporphyrin with considerable hemolysis and a later phase (where ZPP is formed) that
otherwise resembles the common features of lead intoxication. Subacute exposure shows more of
the typical porphyrin response reported with lead. These data may suggest that acute lead
poisoning is quite different from chronic exposure in terms of the nature of hematological
derangement.
Fowler et al. (1980) maintained rats on a regimen of oral lead, starting with exposure of
their dams to lead in water and continuing through 9 months after birth at levels up to
250 ppm lead. The authors observed that the activity of kidney mitochondrial ALA-S and ferro-
chelatase, but not that of the cytosolic enzyme ALA-D, was inhibited. Ferrochelatase activity
was inhibited at 25-, 50-, and 250-ppm exposure levels; activity was 63 percent of the control
values at the 250-ppm level. Depression of state-3 respiration control ratios was observed
for both succinate and pyruvate. Ultrastructurally, the mitochondria were swollen and lyso-
somes were rich in iron. In this study, reduced ferrochelatase activity was observed in
association with mitochondrial injury and disturbance of function. The accumulation of iron
may have been the result of phagocytized dead mitochondria or it may have represented intra-
cellular accumulation of iron, owing to the inability of mitochondria to use the element.
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Ibrahim et al. (1979) have shown that excess intracellular iron under conditions of iron over-
load is stored in cytoplasmic lysosomes. The observation of disturbed mitochondria! respira-
tion suggests, as do the mitochondria! function data of Holtzman and Shen Hsu (1976) and Bull
et a!. (1979) for the developing nervous system, that intramitochondrial transport of iron
would be impaired. Flatmark and Romslo (1975) demonstrated that iron transport in mitochondria
is energy linked and requires an intact respiration chain at the level of cytochrome C, by
which iron (III) on the C-side of the mitochondrial inner membrane is reduced before it is
transported to the M-side and utilized in heme formation.
The above results are particularly interesting in terms of the relative responses of dif-
ferent tissues. While the kidney was affected, there was no change in blood indices of hema-
tological derangement in terms of inhibited ALA-D activity or accumulation of ZPP. This
suggests that there is a difference in dose-effect functions among different tissues, particu-
larly with lead exposure during development of the organism. It appears that while blood
indicators of erythropoietic effects of lead may be more accessible, they may not be the most
sensitive indicators of heme biosynthesis derangement in other organs.
12.3.1.4 Effects of Lead on Coproporphyrin. An increased excretion of coproporphyrin in the
urine of lead workers and children with lead poisoning has long been recognized, and urinary
coproporphyrin measurement has been used as an indicator of lead poisoning. The mechanism of
this enhanced production of coproporphyrin may be direct enzyme inhibition, accumulation of
substrate secondary to inhibition of heme formation, or impaired intramitochondrial movement
of the coproporphyrin. Excess coproporphyrin excretion differs from EP accumulation as an
indicator of lead exposure. The former is a measure of ongoing lead intoxication without the
lag in response seen with EP (Piomelli and Graziano, 1980).
In experimental lead intoxication, there is an accumulation of porphobilinogen and eleva-
ted excretion in urine, owing to inhibition by lead of the enzyme uroporphyrinogen (URO)-I-
synthetase (Piper and Tephly, 1974). In vitro studies of Piper and Tephly (1974) using rat
and human erythrocyte and liver preparations indicate that it is the erythrocyte enzyme
URO-I-synthetase in both rats and humans that is sensitive to the inhibitory effect of lead;
activity of the hepatic enzyme is relatively insensitive. Significant inhibition of the
enzyme's activity occurs at 5 uM lead and virtually total inhibition of activity occurs in
human erythrocyte hemolysates at 10" M. According to Piper and van Lier (1977), the lower
sensitivity of hepatic URO-I-synthetase activity to lead is due to a protective effect
afforded by a pteridine derivative, pteroylpolyglutamate. It appears that the protection does
not occur through lead chelation, since hepatic ALA-D activity was reduced in the presence of
lead. The studies of Piper and Tephly (1974) indicate that it is inhibition of URO-I-
synthetase in erythroid tissue or erythrocytes that accounts for the accumulation of its sub-
strate, porphobilinogen.
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Unlike the case for experimental animals, accumulation of porphobilinogen in plasma and
urine of lead-exposed humans has not been conclusively documented. Absence of porphobilinogen
in urine is a differentiating characteristic in heme biosynthesis disturbance by lead versus
the hepatic porphyrias, acute intermittent porphyrin, and variegate porphyria (Eubanks et
al., 1983).
12.3.2 Effects of Lead on Erythropoiesis and Erythrocyte Physiology
12.3.2.1 Effects of Lead on Hemoglobin Production. Anemia is a manifestation (sometimes an
early one) of chronic lead intoxication. Typically, the anemia is mildly hypochromic and usu-
ally normocytic. It is associated with reticulocytosis, owing to shortened cell survival, and
the irregular presence of basophilic stippling. Its genesis lies in both decreased hemoglobin
production and an increased rate of erythrocyte destruction. Not only is anemia commonly seen
in children with lead poisoning, but it appears to be more severe and frequent among those
with severe lead intoxication (World Health Organization, 1977; National Academy of Sciences,
1972; Lin-Fu, 1973; Betts et al., 1973).
While the anemia associated with lead intoxication in children shows features of iron-
deficiency anemia, there are differences in cases of severe intoxication. These differences
include reticulocytosis, basophilic stippling, and a significantly lower total iron binding
capacity (TIBC). These latter features suggest that iron-deficiency anemia in young children
is exacerbated by lead. The reverse is also true.
In young children, iron deficiency occurs at a significant rate, based on national
(Mahaffey and Michaelson, 1980) and regional (Owen and Lippman, 1977) surveys, and it is known
to be correlated with increased lead absorption in humans (Yip et al., 1981; Chisolm, 1981;
Watson et al. , 1980; Szold, 1974; Watson et al., 1958) and animals (Hamilton, 1978; Barton et
al., 1978; Mahaffey-Six and Goyer, 1972). Hence, prevalent iron deficiency can be seen to
potentiate the effects of lead in reduction of hemoglobin both by increasing lead absorption
and by exacerbating the degree of anemia. Also, in young children, there is a negative corre-
lation between hemoglobin level and blood lead levels (Adebonojo, 1974; Rosen et al., 1974;
Betts et al., 1973; Pueschel et al., 1972). These studies generally involved children under 6
years of age in whom iron deficiency may have been a factor.
In adults, a negative correlation was observed in several studies at blood lead values
usually below 80 pg/dl (Grandjean, 1979; Lilis et al., 1978; Roels et al., 1975a; Wada et
al., 1973), while several studies did not report any relationship below 80 ug/dl (Valentine et
al., 1982; Roels et al., 1979; Ramirez-Cervantes et al., 1978). In adults, iron deficiency
would be expected to play less of a role in this relationship; Lilis et al. (1978) reported
that the significant correlation between lead in blood and hemoglobin level was observed in
workers in whom serum iron and TIBC were indistinguishable from controls.
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The blood lead threshold for effects on hemoglobin has not been conclusively estab-
lished. In children, this value appears to be about 40 ug/dl (World Health Organization,
1977), which is somewhat lower than in adults (Adebonojo, 1974; Rosen et al., 1974; Betts et
al., 1973; Pueschel et al., 1972). Tola et al. (1973) observed no effect of lead on new work-
ers until the blood lead had risen to a value of 50 (jg/dl after about 100 days. The regres-
sion analysis data of Grandjean (1979), Lilis et al. (1978), and Wada et al. (1973) show
persistence of the negative correlation of hemoglobin and blood lead below 50 ug/dl- Human
population dose-response data for the lead-hemoglobin relationship are limited. Baker et al.
(1979) calculated the following percentages of lead workers having a hemoglobin level of
<14.0 g/dl blood at the specified blood lead concentrations: 5 percent at blood lead
values of 40-59 ug/dl; 14 percent at blood lead values of 60-79 ug/dl; and 36 percent at blood
lead values above 80 ug/dl. In 202 lead workers, Grandjean (1979) noted the following per-
centages of workers having a hemoglobin level below 14.4 g/dl at the specified blood lead
concentrations: 17 percent at <25 pg/dl; 26 percent at 25-60 pg/dl; and 45 percent at >60
ug/dl.
The underlying mechanisms of lead-associated anemia appear to be a combination of reduced
hemoglobin production and shortened erythrocyte survival because of direct cell damage. Under
hemoglobin production, biosynthesis of globin, the protein moiety of hemoglobin, appears to be
inhibited as a result of lead exposure (Dresner et al., 1982; Wada et al., 1972; White and
Harvey, 1972; Kassenaar et al., 1957). In the study of White and Harvey (1972), two children
treated for lead intoxication were studied for reticulocyte incorporation of a labeled ami no
acid into alpha and beta globin chains over a post-treatment period when blood lead was
declining. These workers observed that a lag in de novo biosynthesis of alpha versus beta
chains diminished toward a normal ratio (1.0) as blood lead approached 20 ug/dl. These data
are in accord with the observation of Dresner et al. (1982), who noted a reduced globin syn-
thesis (76 percent of controls) in rat bone marrow suspensions exposed to 1.0 uM lead.
Disturbance of globin biosynthesis is a consequence of lead's effects on heme formation
because cellular heme regulates protein synthesis in erythroid cells (Levere and Granick,
1967) and regulates the translation of globin messenger RNA, which may also reflect the effect
of lead on pyrimidine metabolism (Freedman and Rosman, 1976).
12.3.2.2 Effects of Lead on Erythrocyte Morphology and Survival. It is clear that there is a
hemolytic component to lead-induced anemia in humans owing to shortened erythrocyte survival;
the various aspects of this effect have been reviewed by Waldron (1966), Goldberg (1968),
Moore et al. (1980), Valentine and Paglia (1980), and Angle and Mclntire (1982).
The relevant studies of shortened cell life with lead intoxication include observations
of the response of erythrocytes to mechanical and osmotic stress under iji vivo and jri vitro
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conditions. Waldron (1966) has discussed the frequent reports of increased mechanical fragil-
ity of erythrocytes from lead-poisoned workers, beginning with the observations of Aub et al.
(1926). Increased osmotic resistance of erythrocytes from subjects with lead intoxication is
a parallel finding, both jui vivo (Aub and Reznikoff, 1924; Harris and Greenberg, 1954;
Horiguchi et al., 1974) and jjn vitro (Qazi et al., 1972; Waldron, 1964; Clarkson and Kench,
1956). Using an apparatus called a coil planet centrifuge, Karai et al. (1981) studied eryth-
rocytes of lead workers and found significant increases in osmotic resistance; at the same
time, mean corpuscular volume and reticulocyte counts were not different from controls. The
authors suggested that one mechanism of increased resistance involves impairment of hepatic
lecithin-cholesterol acyltransferase, leading to a build-up of cholesterol in the cell mem-
brane. This resembles the increased osmotic resistance seen in obstructive jaundice in which
increased membrane cholesterol has been observed (Cooper et al., 1975). Karai et al. (1981)
also reported an increased cholesterol-phospholipid ratio in lead workers' erythrocytes.
Fukumoto and coworkers (1983) studied the electrophoretic profiles of erythrocyte mem-
brane proteins of a group of lead workers and found that compared to controls there was a
significant negative correlation (r = -0.51, p <0.01) between blood lead and a membrane trans-
fer protein associated with Na and water transport. It appears that one factor in reduced
erythrocyte membrane permeability with lead exposure is a decrease in this protein.
Erythrokinetic data in lead workers and children with lead-associated anemia have been
reported. Shortening of erythrocyte survival has been demonstrated by Hernberg et al. (1967a)
using tritium-labeled difluorophosphonate. Berk et al. (1970) used detailed isotope studies
of a subject with severe lead intoxication to determine shorter erythrocyte life span, while
Leikin and Eng (1963) observed shortened cell survival in three of seven children. These
studies, as well as the reports of Landaw et al. (1973), White and Harvey (1972), Albahary
(1972), and Dagg et al. (1965), indicate that hemolysis is not the exclusive mechanism of ane-
mia and that diminished erythrocyte production also plays a role.
The molecular basis for increased cell destruction with lead exposure includes the inhi-
bition by lead of the activities of the enzymes (Na , K )-ATPase and pyrimidine-5'-nucleoti-
dase (Py-5-N). Erythrocyte membrane (Na , K )-ATPase is a sulfhydryl enzyme and inhibition of
its activity by lead has been well documented (Raghavan et al., 1981; Secchi et al., 1968;
Hasan et al., 1967; Hernberg et al., 1967b). In the study of Raghavan et al. (1981), enzyme
activity was inversely correlated with membrane lead content (p <0.001) in lead workers with
or without symptoms of overt lead toxicity, while correlation with whole blood lead was poor.
With enzyme inhibition, there is irreversible loss of potassium ion from the cell with undis-
turbed input of sodium into the cell, resulting in a relative increase in sodium. Because the
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cells "shrink," there is a net increase in sodium concentration, which likely results in in-
creased mechanical fragility and cell lysis (Moore et al., 1980).
In lead-exposed persons as well as in persons with a genetic deficiency of the enzyme
Py-5-N, reduced activity leads to impaired phosphorolysis of the nucleotides cytidine and
uridine phosphate, which are then retained in the cell and cause interference with the conser-
vation of the purine nucleotides necessary for cellular energetics (Angle and Mclntire, 1982;
Valentine and Paglia, 1980). A more detailed discussion of lead's interaction with this
enzyme is presented in Section 12.3.2.3.
In a series of experiments dealing with the hemolytic relationship of lead and vitamin E
deficiency in animals, Levander et al. (1980) observed that lead exposure exacerbates the
experimental hemolytic anemia associated with vitamin E deficiency by enhancing mechanical
fragility, i.e., by reducing cell deformability. These workers note that vitamin E deficiency
is seen with children having elevated blood lead levels, especially subjects having glucose-6-
phosphate dehydrogenase (G-6-PD) deficiency, indicating that the synergistic relationship seen
in animals may also exist in humans.
Glutathione is a necessary factor in erythrocyte function and structure. In workers ex-
posed to lead, Roels et al. (1975a) found that there is a moderate but significant decrease in
erythrocyte glutathione compared with controls. This appears to reflect lead-induced impair-
ment of glutathione synthesis.
12.3.2.3 Effects of Lead on Pyrimidine-5'-Nuc1eotidase Activity and Erythropoietic Pyrimidine
Metabolism. The presence in lead intoxication of basophilic stippling and an anemia of hemo-
lytic nature is similar to what is seen in subjects having a genetically transmitted defi-
ciency of Py-5-N, an enzyme mediating the phosphorolysis of the pyrimidine nucleotides, cyti-
dine and uridine phosphates. With inhibition, these nucleotides accumulate in the erythrocyte
or reticulocyte, ribonuclease-mediated ribosomal RNA catabolism is retarded in maturing cells,
and the resulting accumulation of aggregates of incompletely degraded ribosomal fragments ac-
counts for the phenomenon of basophilic stippling.
In characterizing the enzyme Py-5-N, Paglia and Valentine (1975) observed that its
activity was particularly sensitive to inhibition by certain metals, particularly lead, promp-
ting further investigation of the interplay between lead intoxication and disturbances of
erythropoietic pyrimidine metabolism. Paglia et al. (1975) observed that in subjects occupa-
tional^ exposed to lead but having no evidence of basophilic stippling or significant fre-
quency of anemia, Py-5-N activity was reduced to about 50 percent of control subjects and was
most impaired (about 11 percent of normal) in one worker with anemia. There was a general
inverse correlation between enzyme activity and blood lead level. In this report, normal
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erythrocytes incubated with varying levels of lead showed detectable inhibition at levels as
low as 0.1-1.0 uM and showed consistent 50 percent inhibition at about 10 uM lead. Subse-
quently, Valentine et al. (1976) observed that an individual with severe lead intoxication had
an 85 percent decrease in Py-5-N activity, basophilic stippling, and accumulation of pyrimi-
dine nucleotides, mainly cytidine triphosphate. Because these parameters approached values
seen in the congenital deficiency of Py-5-N, the data suggest a common etiology for the hemo-
lytic anemia and stippling in both lead poisoning and the congenital disorder.
Several other reports of investigations of Py-5-N activity and pyrimidine nucleotide
levels in lead workers have been published (Paglia et al., 1977; Buc and Kaplan, 1978). In
nine workers having overt lead intoxication and blood lead values of 80-160 ug/dl, Py-5-N
activity was significantly inhibited, and the pyrimidine nucleotides constituted 70-80 percent
of the total nucleotide pool, in contrast to trace levels in unexposed individuals (Paglia
et al., 1977). In the study of Buc and Kaplan (1978), lead workers with or without overt lead
intoxication all showed reduced activity of Py-5-N, which was inversely correlated with blood
lead when the activity was expressed as a ratio with G-6-PD activity to accommodate an en-
hanced population of young cells due to hemolytic anemia. Enzyme inhibition was observed even
when other indicators of lead exposure were negative.
Angle and Mclntire (1978) observed that in 21 children 2-5 years old with blood lead
levels of 7-80 (jg/dl there was a negative linear correlation between Py-5-N activity and blood
lead (r = -0.60, p <0.01). Basophilic stippling was only seen in the child with the highest
blood lead value and only two subjects had reticulocytosis. While adults tended to show a
threshold for inhibition of Py-5-N at a blood lead level of 44 pg/dl or higher, there was no
clear response threshold in these children. In a related investigation with 42 children 1-5
years old with blood lead levels of <10-72 ug/dl, Angle et al. (1982) noted the following: (1)
an inverse correlation (r = -0.64, p <0.001) between the logarithm of Py-5-N activity and
blood lead; (2) a positive log-log correlation between cytidine phosphates and blood lead in
15 of these children (r = 0.89, p <0.001); and (3) an inverse relationship in 12 subjects
between the logarithm of enzyme activity and cytidine phosphates (r = -0.796, p <0.001).
Study of the various relationships at low levels was made possible by the use of anion-
exchange high performance liquid chromatography. In these studies, there was no threshold of
effects of lead on enzyme activity or cell nucleotide content even below 10 ug/dl. Finally,
there was a significant positive correlation of pyrimidine nucleotide accumulation and the
accumulation of ZPP.
In subjects undergoing therapeutic chelation with EDTA, Py-5-N activity increased, while
there was no effect on pyrimidine nucleotides (Swanson et al., 1982). This indicates that the
pyrimidine accumulation was associated with the reticulocyte.
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The metabolic significance of Py-5-N activity inhibition and nucleotide accumulation with
lead exposure is that they affect erythrocyte membrane stability and survival by alteration of
cellular energetics (Angle and Mclntire, 1982). In addition to cell lysis, feedback inhibi-
tion of mRNA and protein synthesis may result through the alteration of globin mRNA or globin
chain synthesis by denatured mRNA. It was noted earlier that disturbances in heme production
also affect the translation of globin mRNA (Freedman and Rosman, 1976). Hence, these two
lead-associated disturbances of erythroid tissue function potentiate the effects of each
other.
12.3.3 Effects of Alkyl Lead on Heme Synthesis and Erythropoiesis
In the discussion of alkyl lead metabolism in Chapter 10, Section 10.7, it was noted that
transformations of tetraethyl and tetramethyl lead iji vivo result in the generation not only
of neurotoxic trialkyl lead metabolites but also products of further dealkylation, including
inorganic lead. One would therefore expect alkyl lead exposure to be associated with, in
addition to other effects, some of those effects classically related to inorganic lead expo-
sure.
Chronic gasoline sniffing has been recognized as a problem habit among children in rural
or remote areas (Boeckx et al., 1977; Kaufman, 1973). When such practice involves leaded gas-
oline, the potential exists for lead intoxication. Boeckx et al. (1977) conducted surveys of
children in remote Canadian communities for the prevalence of gasoline sniffing and indi-
cations of chronic lead exposure. In one group of 43 children who all sniffed gasoline, mean
ALA-D activity was only 30 percent that of control subjects; there was a significant correla-
tion between the decrease in enzyme activity and the frequency of sniffing. A second survey
of 50 children revealed similar results. Two children having acute lead intoxication associ-
ated with gasoline sniffing showed markedly lowered hemoglobin, elevated urinary ALA, and
elevated urinary coproporphyrin. The authors estimated that more than half of the disadvan-
taged children residing in rural or remote areas of Canada may have chronic lead exposure via
this habit; this estimate is consistent with the estimate of Kaufman (1973) of 62 percent for
children in rural American Indian communities in the Southwest.
Robinson (1978) described two cases of pediatric lead poisoning due to habitual gasoline
sniffing, one of which showed basophilic stippling. Hansen and Sharp (1978) reported that a
young adult with acute lead poisioning due to chronic gasoline sniffing not only had basophi-
lic stippling, but a sixfold increase in urinary ALA, elevated urinary coproporphyrin, and an
EP level about fourfold above normal. In the reports of Boeckx et al. (1977) and Robinson
(1978), increased lead levels measured in urine in the course of chelation therapy indicated
that significant amounts of inorganic lead were present.
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12.3.4 The Interrelationship of Lead Effects on Heme Synthesis and the Nervous System
Lead-associated disturbances in heme biosynthesis have been studied as a possible factor
in the neurological effects of lead because of (1) the recognized similarity between classic
signs of lead neurotoxicity and many, but not all, of the neurological components of the gene-
tically transmitted (autosomal dominant) disorder acute intermittent porphyria, and (2) some
unusual aspects of lead neurotoxicity. Both acute porphyria and lead intoxication with neuro-
logical symptoms are variably accompanied by abdominal pain, constipation, vomiting, paralysis
or paresis, demyelination, and psychiatric disturbances (Dagg et al., 1965; Moore et al.,
1980; Silbergeld and Lamon, 1980). According to Angle and Mclntire (1982), some of the
unusual features of lead neurotoxicity are consistent with deranged hematopoiesis: (1) a lag
in production of neurological symptoms; (2) the incongruity of early deficits in affective and
cognitive function with the regional distribution of lead in the brain; and (3) a better
correlation of neurobehavioral deficits with erythrocyte protoporphyrin than with blood lead.
The third feature, it should be noted, is not universally the case (Hammond et al., 1980;
Spivey et al., 1979).
Available evidence points to three specific connections between the heme biosynthetic
pathway and the nervous system in terms of the neurotoxic effects of lead: (1) the potential
neurotoxicity of the heme precursor, ALA; (2) heme deficiency in tissues external to the
nervous system, notably the liver; and (3) jm situ impairment of heme availability in the
nervous system.
While the nature and pattern of the derangements in heme biosynthesis in acute porphyria
and lead intoxication differ in many respects, both involve excessive systemic accumulation
and excretion of ALA, and this common feature has stimulated numerous studies of a connection
between hemato- and neurotoxicity. lt\ vitro data (Whetsell et al., 1978) have shown that the
nervous system is capable of heme biosynthesis in the chick dorsal root ganglion. Sassa et
al. (1979) found that the presence of lead in these preparations increases production of por-
phyrinic material, i.e., there is disturbed heme biosynthesis with accumulation of one or more
porphyrins and, possibly, ALA. Millar et al. (1970) reported inhibited brain ALA-D activity
in suckling rats exposed to lead, while Silbergeld et al. (1982) observed similar inhibition
in brains of adult rats acutely exposed to lead. In the latter study, chronic lead exposure
was also associated with a moderate increase in brain ALA without inhibition of ALA-D, sugges-
ting an extra-neural source of the heme precursor. Finally, Dieter and Finley (1979) showed
marked ALA-D activity depression in brain regions of avian subjects. Moore and Meredith
(1976) administered ALA to rats and observed that exogenous ALA can penetrate the blood-brain
barrier. These reports suggest that ALA can either be generated j_n situ in the nervous system
or can enter the nervous system from elsewhere.
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Neurochemical investigations of ALA action in the nervous system have evaluated interac-
tions with the neurotransmitter gamma-aminobutyric acid (GABA). Interference with GABAergic
function by exposure to lead is compatible with such clinical and experimental signs of lead
neurotoxicity as excitability, hyperactivity, hyperreactivity, and, in severe lead intoxica-
tion, convulsions (Silbergeld and Lamon, 1980). Of particular interest is the similarity in
chemical structures of ALA and GABA; these structures differ only in that ALA has a carbonyl
group on the alpha carbon and GABA has a carbonyl group on the beta carbon.
While chronic lead exposure appears to alter neural pathways involving GABA function
(Silbergeld et al., 1979), this effect cannot be duplicated HI vitro using various levels of
lead (Silbergeld et al., 1980a). This suggests that lead does not impart the effect by direct
interaction or that an intact multi-pathway system is required. In vitro studies (Silbergeld
et al., 1980a; Nicoll, 1976) demonstrate that ALA can displace GABA from synaptosomal mem-
branes associated with synaptic function of the neurotransmitter on the GABA receptor, but
that it is less potent than GABA by a factor of 103-104, suggesting that levels of ALA
achieved even with severe intoxication may not be effectively competitive.
A more significant role for ALA in lead neurotoxicity may well be related to the observa-
tion that GABA release is subject to negative feedback control through presynaptic receptors
on GABAergic terminals (Snodgrass, 1978; Mitchell and Martin, 1978). Brennan and Cantrill
(1979) found that ALA inhibits K+-stimulated release of GABA from preloaded synaptosomes by
functioning as an agonist at the presynaptic receptors. The effect is evident at 1.0 (jM ALA,
and it is abolished by the GABA antagonists bicuculline and picrotoxin. Of interest also is
the demonstration (Silbergeld et al. , 1980a) that synaptosomal release of preloaded 3H-GABA,
both resting and K -stimulated, is also inhibited in animals chronically treated with lead,
paralleling the iji vitro data of Brennan and Cantrill (1979) using ALA.
Silbergeld et al. (1982) described the comparative effects of lead and the agent succi-
nylacetone, given acutely or chronically to adult rats, in terms of disturbances in heme syn-
thesis and neurochemical indices. Succinylacetone, a metabolite that can be isolated from the
urine of patients with hereditary tyrosinemia (Lindblad et al., 1977), is a potent inhibitor
of heme synthesis, exerting its effect by ALA-D inhibition and derepression of ALA synthetase
(Tschudy et al., 1980, 1981). In vivo, both agents showed significant inhibition of high
affinity Na -dependent uptake of 14C-GABA by cortex, caudate, and substantia nigra. However,
neither agent affected GABA uptake jm vitro. Similarly, both chronic and acute lead treatment
and chronically administered succinylacetone reduced the seizure threshold to the GABA antago-
nist, picrotoxin. While these agents may involve entirely different mechanisms of toxicity to
the GABAergic pathway, the fact remains that two distinct potent inhibitors of the heme bio-
synthetic pathway and ALA-D, which do not impart a common neurochemical effect by direct
action on a neurotransmitter function, have a common neurochemical action j_n vivo.
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Several important studies in experimental systems strongly indicate that the key factor
in the connection between heme biosynthesis and neurotoxicity may very well be a reduction in
the levels of heme itself, rather than behavior of its precursor, ALA. Badawy (1978) first
described the role of tryptophan pyrrolase in the relationship of heme biosynthesis and
neurotoxic manifestations of the hepatic porphyrias. Using a porphyric rat model, Litman and
Correia (1983) have reported that lead and the porphyrinic agent DDEP are both associated with
inhibition of the hepatic heme-requiring enzyme system, tryptophan pyrrolase, via reduction of
the free hepatic heme pool. This results not only in elevated plasma tryptophan but also in
significantly elevated brain levels of tryptophan, serotonin, and 5-hydroxyindoleacetic acid.
Of particular interest is the effect of subsequent infusion of heme, which reduced the
elevated levels of these substances to normal amounts. Since, as noted by the authors, heme
does not penetrate the blood-brain barrier, heme repletion had its effect in the liver. This
was confirmed by increases in both hepatic heme content and enzyme activity after heme
infusion. These data are relevant to some of the common features of acute porphyria and
disturbances in tryptophan metabolism noted by Litman and Correia (1983):
(1) Elevated tryptophan levels have been associated with human hepatic encephalo-
pathy.
(2) Elevation in circulating tryptophan in rats produces structural alterations of
brain astrocytes, oligodendroglia, and neurons, as well as Purkinje cell degen-
eration and axonal wasting. These neurohistological changes resemble those
seen in victims of acute porphyria attacks.
(3) The pharmacological effects of serotonin in the central nervous system resemble
the neurological manifestations of acute porphyria attacks.
(4) Administration of tryptophan and serotonin to humans yields symptoms greatly
overlapping those of acute porphyria attacks: psychomotor disturbances,
abdominal pain, nausea, and dysuria.
(5) Porphyric subjects show abnormal tryptophan metabolism and urinary excretion of
large amounts of 5-hydroxyindoleacetic acid.
In the above study, phenobarbital induction of the enzyme system was employed. The
behavior of lead alone has not been investigated. In studies related to heme reduction in the
nervous system itself, Whetsell and Kappas (1981) and Whetsell et al. (1984) showed that co-
administration of heme and lead prevented most of the neuropathic responses to lead in cul-
tured mouse dorsal root ganglion seen with lead alone (see Section 12.3.13). These results
strongly suggest that reduced heme levels in the neural tissue due to the presence of lead are
associated with the adverse effects observed. Because this tissue culture system is known to
12-36
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carry out heme biosynthesis (Whetsell et al., 1978; Sassa et al., 1979), it is highly likely
that lead impairs neural heme biosynthesis.
Human data relating the hemato- and neurotoxicity of lead are limited. Hammond et al.
(1980) reported that the best correlates of the frequency of neurological symptoms in 28 lead
workers were urinary and plasma ALA, as well as blood lead levels, both of which showed a
higher correlation than EP. These data support a connection between heme biosynthesis
impairment and neurological effects of ALA. Of interest here is the clinical report of Lamon
et al. (1979) describing the effect of hematin [Fe(III)-heme] given parenterally to a subject
with lead intoxication. Over the course of treatment (16 days), urinary coproporphyrin and
ALA significantly dropped and neurological symptoms such as lower extremity numbness and
aching diminished. Blood lead levels were not altered during the treatment. Although
remission of symptoms in this subject may have been spontaneous, the outcome parallels that
observed in hematin treatment of subjects with acute porphyria in similar reduction of heme
indicators and relief of symptoms (Lamon et al., 1979).
Taken collectively, all of the available data suggest the following:
(1) Delta-arainolevulinic acid formed jji situ or entering the brain may well £e
neurotoxic by impairing GABAergic function in particular. It inhibits K -
stimulated GABA release by interaction with presynaptic receptors, where ALA
appears to be particularly potent at very low levels (1.0 urn), based on i_n
vitro results.
(2) Decreased levels of heme in the liver due to lead exposure inhibit the activity
of tryptophan pyrrolase, resulting in elevations of tryptophan, serotonin, and
5-hydroxyindoleacetic acid in brain. Such increases are reversed by infusion
of heme.
(3) Heme reduction in neural tissue, as a result of lead's effect on heme biosyn-
thesis, is associated with tissue injury, such injury is prevented by heme co-
administration.
12.3.5 Interference with Vitamin D Metabolism and Associated Physiological Processes
A new dimension to the human toxicology of lead is presented by lead's interaction with
the vitamin D-endocrine system. Recent evidence of lead-induced disturbances in vitamin D
metabolism in humans and animals, particularly with respect to lead-related reductions in the
biosynthesis of the hormonal metabolite 1,25-dihydroxyvitamin D (1,25-(OH)2D), are of special
concern for two reasons: (1) 1,25-(OH)2D appears to serve many more physiological roles than
just mediation of calcium homeostasis and metabolic function, and (2) even moderate levels of
lead exposure in children are associated with vitamin D disturbances that parallel certain
genetic metabolic disorders and other disease states, as well as severe kidney dysfunction.
It appears likely that lead-induced reductions in heme underlie the effects seen in the
vitamin D-endocrine system. This origin would account for the similarities in "thresholds"
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for the effects of lead on both erythrocyte protoporphyrin accumulation and decreases in
levels of 1,25-(OH)2D. It also typifies a cascade of biological effects among various organ
and physiological systems of the body, effects that can ultimately encompass the entire
organism (this is graphically depicted in Chapter 13). Collectively, the interrelationships
of calcium and lead metabolism as well as lead's effects on 1,25-(OH)2D provide one molecular
and mechanistic basis for the classic observation by Aub et al. (1926) that "lead follows the
calcium stream."
12.3.5.1 Relevant Clinical Studies. As initially reported by Rosen et al. (1980), lead in-
toxicated children with blood lead concentrations of 33-120 ug/dl have a marked reduction in
serum levels of 1,25-(OH)2D. The most striking decrease in circulating 1,25-(OH)2D levels was
found in children whose blood lead levels were greater than 62 ug/dl. Nonetheless, highly
significant and profound depressions in circulating 1,25-(OH)2D levels were found also in
children whose blood lead concentrations ranged from 33 to 55 ug/dl. Children whose blood
lead values were above 62 ug/dl also showed a significant decrease in serum total calcium and
ionized calcium (Ca), while serum parathyroid hormone (PTH) concentrations were significantly
elevated. Under these conditions, and in the face of decreased dietary intake of calcium, it
is anticipated that the recognized modulators of 1,25-(OH)2D synthesis (PTH, Ca2+, inorganic
phosphorus [P.]) would enhance production of the vitamin D hormone. Since there was in fact a
reduction in circulating concentrations of 1,25-(OH)2D, this suggests that production of the
vitamin D hormone was actually impaired.
On the basis of significant negative correlations between blood levels of lead and serum
levels of 1,25-(OH)2D and negative correlations between erythrocyte protoporphyrin and 1,25-
(OH)2D in children with blood lead concentrations of 33-120 ug/dl, it is reasonable to
conclude that the lead ion impairs the production of 1,25-(OH)2D3, as aluminum does in chil-
dren undergoing total parenteral nutrition (Rosen and Chesney, 1983).
The 1-hydroxylation step to produce the vitamin 0 hormone is carried out in the mitochon-
dria of the renal tubule by a complex cytochrome P-450 enzyme system (Rosen and Chesney,
1983). The ingredients of this enzyme system include intact mitochondria, Krebs cycle sub-
strates, cytochrome P-450 electron transport, oxidative phosphorylation, and generation of
NADPH (nicotinamide adenine dinucleotide phosphate, reduced). The biosynthesis of the vita-
min D hormone is controlled in large part by the functional integrity of mitochondria, by the
ionic (Ca, P.) microenvironment of the extracellular fluid, and by the uptake of calcium by
mitochondria (including the delicate homeostasis characteristic of intracellular calcium con-
centrations and calcium pumps). It is clear, therefore, based upon lead's toxic effects on
mitochondria, cellular energetics, and cytochrome P-450 electron transport in several studies,
including some on children (Saenger et al., 1984; Piomelli et al. , 1982), that lead most
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likely impairs the 1-hydroxylase enzyme system, although altered peripheral metabolism of
1,25-(OH)2D cannot be completely ruled out. As noted in Section 12.5, furthermore, lead inhi-
bits renal ferrochelatase with accumulation of EP leading to a reduction in the kidney heme
pool and reduced availability of heme for renal 1-hydroxylase (Fowler et al., 1980).
Lead's impairment of the 1-hydroxylase enzyme system in lead intoxicated children is
strongly supported by additional information: 1) 1,25-(OH)2D levels in serum returned to
normal values within two days following chelation therapy, while no changes were found in
25-(OH)D levels, and 2) a strong negative correlation between 1,25-(OH)2D values and blood
lead was found over the entire range (12-120 ug/dl) of blood lead levels measured in the study
(Rosen etal., 1980; Mahaffey etal., 1982). Furthermore, no change in the slope of the
regression line between 1,25-(OH)2D and blood lead was found for blood lead values above or
below 30 ug/dl (Mahaffey et al. , 1982). These findings provide considerable support for the
view that lead interferes with normal ionic transport in cells and with the functional integ-
rity of mitochondria that carry out this 1-hydroxylation. In terms of ionic transport, cur-
rent information indicates that increasing extracellular and intracellular concentrations of
calcium depress production of the vitamin D hormone (Rosen and Chesney, 1983). If renal
tubule cells accumulate high concentrations of calcium after exposure to lead, as do hepato-
cytes (Pounds et al., 1982a, Pounds and Mittelstaedt, 1983), osteoclasts (Rosen, 1983, 1985),
and brain cells (Silbergeld and Adler, 1978), renal tubule cells may consequently "turn off"
1,25-(OH)2D production. Such an effect is likely to be reversible when lead is decreased in
the extracellular fluid, as it is in children after therapy with CaNa2EDTA.
In summary, lead's effect(s) on the complex 1-hydroxylase enzyme system may be expressed
in one or several components of the enzyme (e.g., cellular energetics, integrity of mitochon-
dria). Simultaneously, lead may interfere with ionic regulation of 1,25-(OH)2D3 biosynthesis.
The fact that such effects can be reversed, at least insofar as 1,25-(OH)2D levels may recover
to normal values after chelation therapy, does not suggest that these effects of lead are
necessarily transient or subject to physiological adaptation. Thus far, reversibility has
been known to occur only after medical intervention.
12.3.5.2 Experimental Studies. Smith et al. (1981) observed depressions of plasma
1,25-(OH)2D in rats fed 0.82 percent lead as lead acetate. Moreover, lead ingestion totally
blocked the intestinal calcium transport response to the vitamin D hormone. Though the dose
of lead and the resulting blood lead concentrations were high in this study, it confirms the
effect(s) of lead on vitamin D metabolism reported in children. A recent study demonstrated
directly that renal production and tissue levels of the vitamin 0 hormone were reduced in a
dose-related fashion in chicks fed a diet supplemented with lead (Edelstein etal., 1984).
Previous studies have shown that vitamin D and 1,25-(OH)2D3 enhance lead acetate absorption in
the distal small intestine of the rat, whereas vitamin D-dependent calcium absorption occurs
12-39
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in the proximal duodenum (Smith et al., 1978; Mahaffey et al., 1979). It is likely that
vitamin D affects lead absorption in a manner somewhat different from the manner in which it
affects calcium absorption (Smith et al., 1978; Mykkanen and Wasserman, 1982). It is of
interest that high doses of vitamin D and 1,25-(OH)2D3 do not markedly increase lead absorp-
tion above that achieved with physiological doses (Smith et al., 1978).
12.3.5.3 Implications of Lead Effects on Vitamin D Metabolism
12.3.5.3.1 Direct Metabolic Consequences of Vitamin D Metabolism Interference in Children. A
lower daily intake of calcium, as observed in lead intoxicated children (Sorrell et al. , 1977;
Rosen et al., 1980), accompanied by a relative decrease in l,25-(OH)2D-stimulated formation of
calcium-binding protein (CaBP), may permit lead to compete favorably with calcium for mucosal
proteins and similar absorption sites in the intestine. In addition, experimental study with
animal CaBP has demonstrated a much greater affinity of this intestinal protein for lead than
for calcium (Fullmer et al., 1985). Such findings help explain the negative correlations
found between calcium intake and blood lead and between serum calcium and blood lead values
(Sorrell et al., 1977).
Furthermore, depression in serum ionized calcium during lead intoxication (Rosen et al.,
1980) may enhance the movement of lead from hard tissue to critical organ sites in soft
tissues. In bone organ culture, decreasing the concentration of calcium in the medium
enhances mobilization of previously incorporated radioactive lead from bone explants to the
medium (Rosen and Markowitz, 1980). Among other things, these findings indicate that reduced
1,25-(OH)2D levels do not serve to "protect" soft target organs such as brain and kidney from
lead deposition by sequestering the metal in bone. Further, empirical support for this con-
clusion may be found in the results of Smith et al. (1981), who reported no consistent differ-
ences in rats' kidney lead content as a function of the presence or absence of a vitamin D
supplement in the diet. These investigators also found no significant differences in the
blood lead concentrations of the rats as a function of vitamin D supplementation. In summary,
there is little reason to suppose that reduced levels of 1,25-(OH)2D might function as part of
a negative feedback process to reduce further absorption of lead or to mitigate its toxic
effects on various target organs.
12.3.5.3.2 Other Childhood Diseases Associated with a Reduction in Circulating 1.25-(OH)?D
as a Reflection of Depressed Biosynthesis. At blood lead levels of 33-55 ug/dl (Rosen et al.,
1980), 1,25-(OH)2D levels are reduced to levels comparable to those observed in children who
have severe renal insufficiency with loss of about two-thirds of their normal renal function
(Rosen and Chesney, 1983; Chesney et al., 1983). Also, at blood lead levels of 33-120 ug/dl,
analogous depressions in 1,25-(OH)2D concentrations (S20 pg/ml) are found in:
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(1) Vitamin D-dependent rickets, type I--an inborn error of vitamin D metabolism in
which the 1-hydroxylase enzyme system (or a component thereof) is virtually
absent;
(2) Oxalosis—an inborne error of metabolism in which calcium oxalate crystals
are deposited throughout the body, including the kidney, and result in chronic
renal insufficiency,
(3) Hormone-deficient hypoparathyroidi sin—thought to be an autoimmune disorder,
hereditary in some cases, that is characterized by parathyroid hormone defi-
ciency and, as a result, decreased production of the vitamin D hormone;
(4) Aluminum intoxication in children undergoing total parenteral nutrition—has
occurred when the casein hydrolysates used were contaminated with aluminum.
These disorders are reviewed by Rosen and Chesney (1983) and Chesney et al. (1983).
12.3.5.3.3 Physiological Functions of 1,25-(OH)2D3 at the Cellular Level. The vitamin D-
endocrine system is responsible in large part for the maintenance of extra- and intracellular
calcium homeostasis (Rasmussen and Waisman, 1983; Wong, 1983; Shlossman etal., 1982; Rosen
and Chesney, 1983). As a result, the integrity of cells of diverse function is preserved, as
are numerous calcium-mediated functions. It is known that calcium, an important participant
in the hormonal responses of many target cell systems (Rasmussen and Waisman, 1983), acts not
only as a second messenger, but also as a modulator of cyclic nucleotide metabolism. The tem-
poral and spatial regulation of cellular calcium is exceedingly important in the response of a
variety of cells to hormonal and electrical stimuli.
Lead alone (without hormones) produces an overamplification of calcium influx in hepato-
cytes (Pounds etal., 1982a), osteoclasts (Rosen, 1983, 1985), and brain slices (Silbergeld
and Adler, 1978) at relatively low concentrations. As a result, calcium-mediated cell func-
tions are perturbed (Pounds et al., 1982b). Based upon these findings, it is reasonable to
conclude that modulation in cellular calcium metabolism induced by lead at relatively low
concentrations may have the potential of disturbing multiple functions of different tissues
that depend upon calcium as a second messenger. Perturbations in cellular calcium homeostasis
may thereby result from the effects of lead alone; but these effects may be enhanced when
coupled with decreased production of 1,25-(OH)2D3 and reduction in serum (and extracellular
fluid) ionized calcium values observed in lead intoxicated children.
Calmodulin is of central importance as an intracellular calcium receptor protein. Its
nearly universal distribution in mammalian cells emphasizes further that calcium serves as a
"universal" second messenger. As such, calmodulin regulates several enzyme systems and trans-
port processes. The list of calcium-sensitive reactions modulated by the calmodulin-calcium
complex is rapidly expanding (see review by Cheung, 1980). Recently, it has been shown that
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lead can replace calcium in the activation of calmodulin-sensitive processes (Habermann et
al., 1983), including potassium loss from erythrocytes (Goldstein and Ar, 1983). Though at an
early stage of investigation, it is conceivable that a molecular model of lead toxicity may
include (in addition to those processes cited above) intracellular occupation of calcium-
binding sites on calmodulin. Since calmodulin regulates multiple cell activities, such a
mechanism may underlie some of the diverse effects of lead. Lead alone and lead's inter-
action^) with calmodulin and intracellular calcium homeostasis are inherently coupled to the
vitamin D-endocrine system.
12.3.5.3.4 Cell Differentiation/Maturation. Tumor cell lines possess cytosol receptors to
which 1,25-(OH)2D3 binds specifically (Tanaka et al., 1982; Shiina et al., 1983; Honma et al.,
1983). Human promyelocytic leukemia cells (HL-60) can be induced to differentiate rn vitro by
1,25-(OH)2D3. Differentiation-associated properties, such as phagocytosis and C3 rosette
formation, were induced by as little as 0.12 nM 1,25-(OH)2D3. As cells exhibited differentia-
tion, the viable cell number was decreased to less than half of the control (Tanaka et al.,
1982). A specific cytosol protein that bound 1,25-(OH)2D3 was found in these HL-60 cells; its
physical and biochemical properties closely resembled those found in "classical" vitamin D
target tissues. These and other studies noted above indicated that 1,25-(OH)2D3 induced
differentiation of HL-60 cells by a mechanism similar to that proposed for the classical con-
cept of steroid hormone action. This common mechanism of steroid hormone action includes
binding of hormone to a cytosol receptor (to form a hormone-receptor complex) and subsequent
movement of this complex into the nucleus where it binds to chromatin.
A recent study by Honma et al. (1983) showed that the survival time of syngeneic SL mice
inoculated with murine myeloid leukemia cells (ML) was markedly prolonged by l-25-(OH)2D3
treatment (12.5-50 pmol per mouse). Evidence indicated that induction of differentiation of
ML cells into macrophages jn vitro was correlated with its effect in prolonging survival time;
and it was suggested that the role of 1,25-(OH)2D3 in decreasing leukemogenicity of ML cells
HI vivo is due to its effect in suppressing proliferation and inducing differentiation of ML
cells iji vitro (Honma et al., 1983).
It is evident, therefore, that the differentiation in HL-60 cells (and other cell lines)
caused by 1,25-(OH)2D3 is a manifestation of the normal action of this hormone to elicit
maturation of myeloid stem cells into macrophages. Because macrophages are thought to be the
precursor of bone resorbing osteoclasts, this is a logical mechanism whereby 1,25-(OH)2D3
brings about calcium resorption/homeostasis through recruitment of cells competent in bone re-
modeling. Moreover, parathyroid hormone is thought to act both directly to stimulate osteo-
clast production from myeloid precursors and on T-lymphocytes to cause the elaboration of
putative osteoclast-enhancing factors. It appears, therefore, that the vitamin 0 hormone
regulates calcium homeostasis and also participates directly in bone turnover by orchestrating
the population of cells within bone.
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12.3.5.3.5 Immunoregulatory Role of the Vitamin D Hormone. The widespread distribution of
receptors for 1,25-(OH)2D3 in tissues not thought to play a role in mineral metabolism has
made it clear that the vitamin D hormone plays a wider biologic role than was previously
thought (Kadowaki and Norman, 1984a,b; Stumpf et al., 1982; Clark et al., 1981; Gel bard et
a!., 1980). Receptors for 1,25-(OH)2D3 are present in normal human monocytes and malignant
lymphocytes (Provvedini et al., 1983; Bhalla et al. , 1983). It has also been shown that
macrophages from vitamin D-deficient mice have impaired phagocytic and inflammatory responses
correctable by 1,25-(OH)2D3 repletion (Bar-Shavit et al., 1981).
T and B lymphocytes obtained from normal humans also expressed the 1,25-(OH)2D3 receptor
after the lymphocytes had been activated by mitogenic lectins and Epstein-Barr virus
(Provvedini et al., 1983; Bhalla et al., 1983). The mitogenic lectin phytohemagglutinin (PHA)
stimulates T lymphocyte proliferation and induces the production of various lymphokines,
including interleukin-2 (IL-2), which is important for the growth of T cells. Recently, it
has been demonstrated that 1,25-(OH)2D3 (at picomolar concentrations) inhibits the growth-
promoting lymphokine IL-2 and the proliferation of PHA-stimulated lymphocytes obtained from
normal humans (Tsoukas et al., 1984). These results confirm and extend earlier evidence that
1,25-(OH)2D3 receptors are expressed in T lymphocytes activated with mitogenic lectins
(Provvedini et al., 1983; Bhalla et al., 1983). In light of suggestions that calcium translo-
cation is involved in the mitogen-induced activation of lymphocytes and in view of the well-
recognized calcitropic effects of 1,25-(OH)2D3 on mineral-dependent target tissues, it may be
that the suppressive effect of the vitamin D hormone on IL-2 is mediated by calcium transloca-
tion (Tsoukas et al., 1984). However mediated, this effect demonstrates the immunoregulatory
role of 1,25-(OH)2D3 and, thus, another possible means by which lead could affect immunity
(see Section 12.8).
12.3.6 Summary and Overview
12.3.6.1 Effects of Lead on Heme Biosynthesis. The effects of lead on heme biosynthesis are
well known because of their clinical prominence and the numerous studies of such effects in
humans and experimental animals. The process of heme biosynthesis starts with glycine and
succinyl-coenzyme A, proceeds through formation of protoporphyrin IX, and culminates with the
insertion of divalent iron into the porphyrin ring to form heme. In addition to being a con-
stituent of hemoglobin, heme is the prosthetic group of many tissue hemoproteins having vari-
able functions, such as myoglobin, the P-450 component of the mixed-function oxygenase system,
and the cytochromes of cellular energetics. Hence, disturbance of heme biosynthesis by lead
poses the potential for multiple-organ toxicity.
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In investigations of lead's effects on the heme synthesis pathway, most attention has
been devoted to the following: (1) stimulation of mitochondrial delta-aminolevulinic acid
synthetase (ALA-S), which mediates formation of delta-aminolevulinic acid (ALA); (2) direct
inhibition of the cytosolic enzyme, delta-aminolevulinic acid dehydrase (ALA-D), which cata-
lyzes formation of porphobilinogen from two units of ALA; and (3) inhibition of insertion of
iron (II) into protoporphyrin IX to form heme, a process mediated by ferrochelatase.
Increased ALA-S activity has been found in lead workers as well as in lead-exposed ani-
mals, although an actual decrease in enzyme activity has also been observed in several experi-
mental studies using different exposure methods. It appears, then, that the effect on ALA-S
activity may depend on the nature of the exposure. Using rat liver cells in culture, ALA-S
activity was stimulated jji vitro at lead levels as low as 5.0 uM or 1.0 ug/g preparation. The
increased activity was due to biosynthesis of more enzyme. The blood lead threshold for stim-
ulation of ALA-S activity in humans, based on a study using leukocytes from lead workers,
appears to be about 40 ug/dl. Whether this apparent threshold applies to other tissues de-
pends on how well the sensitivity of leukocyte mitochondria mirrors that in other systems.
The relative impact of ALA-S activity stimulation on ALA accumulation at lower lead exposure
levels appears to be much less than the effect of ALA-D activity inhibition. ALA-D activity
is significantly depressed at 40 pg/dl blood lead, the point at which ALA-S activity only
begins to be affected.
Erythrocyte ALA-D activity is very sensitive to inhibition by lead. This inhibition is
reversed by reactivation of the sulfhydryl group with agents such as dithiothreitol, zinc, or
zinc and glutathione. Zinc levels that achieve reactivation, however, are well above physio-
logical levels. Although zinc appears to offset the inhibitory effects of lead observed in
animal studies and in human erythrocytes jin vitro, lead workers exposed to both zinc and lead
do not show significant changes in the relationship of ALA-D activity to blood lead when com-
pared with workers exposed just to lead. Nor does the range of physiological zinc levels in
nonexposed subjects affect ALA-D activity. In contrast, zinc deficiency in animals signifi-
cantly inhibits ALA-D activity, with concomitant accumulation of ALA in urine. Because zinc
deficiency has also been demonstrated to increase lead absorption, the possibility exists for
the following dual effects of such deficiency on ALA-D activity: (1) a direct effect on acti-
vity due to reduced zinc availability; and (2) increased lead absorption leading to further
inhibition of activity.
Erythrocyte ALA-D activity appears to be inhibited at virtually all blood lead levels
measured so far, and any threshold for this effect in either adults or children remains to be
determined. A further measure of this enzyme's sensitivity to lead is a report that rat bone
marrow suspensions show inhibition of ALA-D activity by lead at a level of 0.1 ug/g suspen-
sion. Inhibition of ALA-D activity in erythrocytes apparently reflects a similar effect in
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other tissues. Hepatic ALA-D activity in lead workers was inversely correlated with erythro-
cyte activity as well as blood lead levels. Of significance are experimental animal data
showing that (1) brain ALA-D activity is inhibited with lead exposure, and (2) this inhibition
appears to occur to a greater extent in developing animals than in adults, presumably reflec-
ting greater retention of lead in developing animals. In the avian brain, cerebellar ALA-D
activity is affected to a greater extent than that of the cerebrum and, relative to lead con-
centration, shows inhibition approaching that occurring in erythrocytes.
Inhibition of ALA-D activity by lead is reflected by elevated levels of its substrate,
ALA, in blood, urine, and soft tissues. Urinary ALA is employed extensively as an indicator
of excessive lead exposure in lead workers. The diagnostic value of this measurement in pedi-
atric screening, however, is limited when only spot urine collection is done; more satisfac-
tory data are obtainable with 24-hr collections. Numerous independent studies document a
direct correlation between blood lead and the logarithm of urinary ALA in human adults and
children; the blood lead threshold for increases in urinary ALA is commonly accepted as 40
pg/dl. However, several studies of lead workers indicate that the correlation between urinary
ALA and blood lead continues below this value; one study found that the slope of the dose-
effect curve in lead workers depends on the level of exposure.
The health significance of lead-inhibited ALA-D activity and accumulation of ALA at lower
lead exposure levels is controversial. The "reserve capacity" of ALA-D activity is such that
only the level of inhibition associated with marked accumulation of the enzyme's substrate,
ALA, in accessible indicator media may be significant. However, it is not possible to quan-
tify at lower levels of lead exposure the relationship of urinary ALA to target tissue levels
or to relate the potential neurotoxicity of ALA at any accumulation level to levels in indi-
cator media. Thus, the blood lead threshold for neurotoxicity of ALA may be different from
that associated with increased urinary excretion of ALA.
Accumulation of protoporphyrin in erythrocytes of lead-intoxicated individuals has been
recognized since the 1930s, but it has only recently been possible to quantitatively assess
the nature of this effect via development of sensitive, specific microanalysis methods. Accu-
mulation of protoporphyrin IX in erythrocytes results from impaired placement of iron (II) in
the porphyrin moiety in heme formation, an intramitochondrial process mediated by ferrochela-
tase. In lead exposure, the porphyrin acquires a zinc ion in lieu of native iron, thus form-
ing zinc protoporphyrin (ZPP), which is tightly bound in available heme pockets for the life
of the erythrocytes. This tight sequestration contrasts with the relatively mobile nonmetal,
or free, erythrocyte protoporphyrin (FEP) accumulated in the congenital disorder erythropoiet-
ic protoporphyria.
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Elevation of erythrocyte ZPP has been extensively documented as exponentially correlated
with blood lead in children and adult lead workers and is currently considered one of the best
indicators of undue lead exposure. Accumulation of ZPP only occurs in erythrocytes formed
during lead's presence in erythroid tissue; this results in a lag of at least several weeks
before its buildup can be measured. The level of ZPP accumulation in erythrocytes of newly
employed lead workers continues to increase after blood lead has already reached a plateau.
This influences the relative correlation of ZPP and blood lead in workers with short exposure
histories. Also, the ZPP level in blood declines much more slowly than blood lead, even after
removal from exposure or after a drop in blood lead. ZPP level also appears to be a more re-
liable indicator of continuing intoxication from lead resorbed from bone in former lead
workers long removed from heavy lead exposure.
The threshold for detection of lead-induced ZPP accumulation is affected by the relative
spread of blood lead and corresponding ZPP values measured. In young children (<4 yr old),
the ZPP elevation associated with iron-deficiency anemia must also be considered. In adults,
numerous studies indicate that the blood lead threshold for ZPP elevation is about 25-30
ug/dl. In children 10-15 years old, the threshold is about 16 ug/dl; for this age group, iron
deficiency is not a factor. In one study, children over 4 years old showed the same thresh-
old, 15.5 ug/dl, as a second group under 4 years old, indicating that iron deficiency was not
a factor in the study. At 35.2 ug/dl blood lead, 50 percent of the children had significantly
elevated FEP levels (2 standard deviations above the reference mean FEP).
At blood lead levels below 30-40 ug/dl, any assessment of the EP-blood lead relationship
is strongly influenced by the relative analytical proficiency of measurements of both blood
lead and EP. The types of statistical analyses used are also important. In a recent detailed
statistical study involving 2004 children, 1852 of whom had blood lead values below 30 ug/dl,
segmental line and probit analysis techniques were employed to assess the dose-effect thres-
hold and dose-response relationship. An average blood lead threshold for the effect using
both statistical techniques was 16.5 ug/dl for the full group and for those subjects with
blood lead below 30 (jg/dl• The effect of iron deficiency was tested for and was removed. Of
particular interest was the finding that blood lead values of 28.6 and 35.2 ug/dl corresponded
to EP elevations of more than 1 or 2 standard deviations, respectively, above the reference
mean in 50 percent of the children. Hence, fully half of the children had significant ele-
vations of EP at blood lead levels around 30 ug/dl. From various reports, children and adult
females appear to be more sensitive to lead's effects on EP accumulation at any given blood
lead level; children are somewhat more sensitive than adult females.
Lead's effects on heme formation are not restricted to the erythropoietic system. Recent
data indicate that the reduction of serum 1,25-dihydroxyvitamin D seen with even low-level
lead exposure is apparently the result of lead-induced inhibition of the activity of renal
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1-hydroxylase, a cytochrome P-450 mediated enzyme. Moreover lead inhibits renal ferrochela-
tase activity, which, with elevated kidney EP, leads to a reduction of heme available for
heme-requiring enzymes such as renal 1-hydroxylase. Reduction in activity of the hepatic
enzyme tryptophan pyrrolase and concomitant increases in plasma tryptophan as well as brain
tryptophan, serotonin, and hydroxyindoleacetic acid have been shown to be associated with
lead-induced reduction of the hepatic heme pool. The heme-contaim'ng hepatic protein cyto-
chrome P-450 (an integral part of the hepatic mixed-function oxygenase system) is affected in
humans and animals by lead exposure, especially acute intoxication. Reduced P-450 content
correlates with impaired activity of detoxifying enzyme systems such as aniline hydroxylase
and aminopyrine demethylase. It is also responsible for reduced 6p-hydroxylation of cortisol
in children having moderate lead exposure.
Studies of organotypic chick and mouse dorsal root ganglion in culture show that the ner-
vous system has heme biosynthetic capability and that not only is this capability reduced in
the presence of lead but production of porphyrinic material is increased. In the neonatal
rat, depending on the age at dosing and the duration of dosing, chronic lead exposure result-
ing in moderately elevated blood lead is associated with retarded increases in the hemoprotein
cytochromes and with disturbed electron transport in the developing cerebral cortex. These
data parallel effects of lead on ALA-D activity and ALA accumulation in neural tissue. When
both of these effects are viewed in the toxicokinetic context of increased retention of lead
in both developing animals and children, there is an obvious and serious potential for
impaired heme-based metabolic function in the nervous system of lead-exposed children.
As can be concluded from the above discussion, the health significance of ZPP accumula-
tion rests with the fact that it is evidence of impaired heme and hemoprotein formation in
many tissues that arises from entry of lead into mitochondria. Elevation of EP in children at
relatively low blood lead levels is considered by the pediatric medicine community to be a
matter of concern, and the Centers for Disease Control in their recent statement on lead poi-
soning in children (U.S. Centers for Disease Control, 1985) have noted that a blood lead level
above 25 ug/dl along with an EP level above 35 ug/dl whole blood is to be taken as early evi-
dence of lead toxicity. Such evidence for reduced heme synthesis is consistent with a great
deal of data documenting lead-associated effects on mitochondria. The relative value of the
lead-ZPP relationship in erythropoietic tissue as an index of this effect in other tissues
hinges on the relative sensitivity of the erythropoietic system compared with other organ
systems. One study of rats exposed over their lifetime to low levels of lead demonstrated
that protoporphyrin accumulation in renal tissue was already significant at levels of lead
exposure which produced little change in erythrocyte porphyrin levels.
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Other steps in the heme biosynthesis pathway are also known to be affected by lead, al-
though these have not been as well studied on a biochemical or molecular level. Coproporphy-
rin levels are increased in urine, reflecting active lead intoxication. Lead also affects the
activity of the enzyme uroporphyrinogen-I-synthetase in experimental animal systems, resulting
in an accumulation of its substrate, porphobilinogen. The erythrocyte enzyme has been re-
ported to be much more sensitive to lead than the hepatic species, presumably accounting for
much of the accumulated substrate. Unlike the case with experimental animals, lead-exposed
humans show no rise in urinary porphobilinogen, which is a differentiating characteristic of
lead intoxication versus the hepatic porphyrias. Ferrochelatase is an intramitochondrial
enzyme, and impairment of its activity either directly by lead or via impairment of iron
transport to the enzyme is evidence of the presence of lead in mitochondria.
12.3.6.2 Lead Effects on Erythropoiesis and Erythrocyte Physiology. Anemia is a manifesta-
tion of chronic lead intoxication and is characterized as mildly hypochromic and usually nor-
mocytic. It is associated with reticulocytosis, owing to shortened cell survival, and the
variable presence of basophilic stippling. Its occurrence is due to both decreased production
and increased rate of destruction of erythrocytes. In young children (<4 yr old), iron defi-
ciency anemia is exacerbated by lead uptake, and vice versa. Hemoglobin production is nega-
tively correlated with blood lead in young children, in whom iron deficiency may be a con-
founding factor, as well as in lead workers. In one study, blood lead values that were
usually below 80 (jg/dl were inversely correlated with hemoglobin content. In these subjects
no iron deficiency was found. The blood lead threshold for reduced hemoglobin content is
about 50 pg/dl in adult lead workers and somewhat lower (about 40 ug/dl) in children.
The mechanism of lead-associated anemia appears to be a combination of reduced hemoglobin
production and shortened erythrocyte survival due to direct cell injury. Lead's effects on
hemoglobin production involve disturbances of both heme and globin biosynthesis. The hemoly-
tic component to lead-induced anemia appears to be caused by increased cell fragility and in-
creased osmotic resistance. In one study using rats, the hemolysis associated with vitamin E
deficiency, via reduced cell deformability, was exacerbated by lead exposure. The molecular
basis for increased cell destruction rests with inhibition of (Na , K )-ATPase and pyrimi-
dine-5'-nucleotidase. Inhibition of the former enzyme leads to cell "shrinkage" and inhibi-
tion of the latter results in impaired pyrimidine nucleotide phosphorolysis and disturbance of
the activity of the purine nucleotides necessary for cellular energetics.
12.3.6.3 Effects of lead on erythropoietic pyrimidine metabolism. In lead intoxication, the
presence of both basophilic stippling and anemia with a hemolytic component is due to inhibi-
tion by lead of the activity of pyrimidine-5'-nucleotidase (Py-5-N), an enzyme that mediates
the dephosphorylation of pyrimidine nucleotides in the maturing erythrocyte. Inhibition of
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this enzyme by lead has been documented in lead workers, lead-exposed children, and experimen-
tal animal models. In one study of lead-exposed children, there was a negative correlation
between blood lead and enzyme activity, with no clear response threshold. A related report
noted that, in addition, there was a positive correlation between cytidine phosphate and blood
lead and an inverse correlation between pyrimidine nucleotide and enzyme activity.
The metabolic significance of Py-5-N inhibition and cell nucleotide accumulation is that
they affect erythrocyte stability and survival as well as potentially affect mRNA and protein
synthesis related to globin chain synthesis. Based on one study of children, the threshold
for the inhibition of Py-5-N activity appears to be about 10 ug/dl blood lead. Lead's inhibi-
tion of Py-5-N activity and a threshold for such inhibition are not by themselves the issue.
Rather, the issue is the relationship of such inhibition to a significant level of impaired
pyrimidine nucleotide metabolism and the consequences for erythrocyte stability and function.
The relationship of Py-5-N activity inhibition by lead to accumulation of its pyrimidine
nucleotide substrate is analogous to lead's inhibition of ALA-D activity and accumulation of
ALA.
12.3.6.4 Effects of Alkyl Lead Compounds on Heme Biosynthesis and Erythropoiesis. Tetraethyl
lead and tetramethyl lead, components of leaded gasoline, undergo transformation iji vivo to
neurotoxic trialkyl metabolites as well as further conversion to inorganic lead. Hence, one
might anticipate that exposure to such agents may result in effects commonly associated with
inorganic lead, particularly in terms of heme synthesis and erythropoiesis. Various surveys
and case reports show that the habit of sniffing leaded gasoline is associated with chronic
lead intoxication in children from socially deprived backgrounds in rural or remote areas.
Notable in these subjects is evidence of impaired heme biosynthesis, as indexed by signifi-
cantly reduced ALA-D activity. In several case reports of frank lead toxicity from habitual
leaded gasoline sniffing, effects such as basophilic stippling in erythrocytes and signifi-
cantly reduced hemoglobin have also been noted.
12.3.6.5 Relationships of Lead Effects on Heme Synthesis to Neurotoxicity. The role of lead-
associated disturbances of heme biosynthesis as a possible factor in neurological effects of
lead is of considerable interest due to the following: (1) similarities between classical
signs of lead neurotoxicity and several neurological components of the congenital disorder
acute intermittent porphyria; and (2) some of the unusual aspects of lead neurotoxicity.
There are three possible points of connection between lead's effects on heme biosynthesis and
the nervous system. Associated with both lead neurotoxicity and acute intermittent porphyria
is the common feature of excessive systemic accumulation and excretion of ALA. In addition,
lead neurotoxicity reflects, to some degree, impaired synthesis of heme and hemoproteins in-
volved in crucial cellular functions; such an effect on heme is now known to be relevant
within neural tissue as well as in non-neural tissue.
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Available information indicates that ALA levels are elevated in the brains of lead-
exposed animals and arise through in situ inhibition of brain ALA-D activity or through trans-
port of ALA to the brain after formation in other tissues. ALA is known to traverse the
blood-brain barrier. Hence, ALA is accessible to, or formed within, the brain during lead
exposure and may express its neurotoxic potential.
Based on various ir\ vitro and j_n vivo neurochemical studies of lead neurotoxicity, it
appears that ALA can inhibit release of the neurotransmitter gamma-aminobutyric acid (GABA)
from presynaptic receptors at which ALA appears to be very potent even at low levels. In an
_in vitro study, agonist behavior by ALA was demonstrated at levels as low as 1.0 UM ALA. This
HI vitro observation supports results of a study using lead-exposed rats in which there was
inhibition of both resting and K -stimulated release of preloaded 3H-GABA from nerve termi-
nals. The observation that iji vivo effects of lead on neurotransmitter function cannot be
duplicated with uj vitro preparations containing added lead is further evidence of an effect
of some agent (other than lead) that acts directly on this function. Human data on lead-
induced associations between disturbed heme synthesis and neurotoxicity, while limited, also
suggest that ALA may function as a neurotoxicant.
A number of studies strongly suggest that lead-impaired heme production itself may be a
factor in the toxicant's neurotoxicity. In porphyric rats treated also with phenobarbital,
both lead and the organic agent DDEP inhibit tryptophan pyrrolase activity owing to reductions
in the hepatic heme pool, thereby leading to elevated levels of tryptophan and serotonin in
the brain. Such elevations are known to induce many of the neurotoxic effects also seen with
lead exposure. Of great interest is the fact that heme infusion in these animals reduces
brain levels of these substances and also restores enzyme activity and the hepatic heme pool.
It remains to be demonstrated that use of lead alone, without enzyme induction, would show
similar effects. Another line of evidence for the heme-basis of lead neurotoxicity is that
mouse dorsal root ganglion in culture manifests morphological evidence of neural injury with
rather low lead exposure, but such changes are largely prevented with co-administration of
heme. Finally, studies also show that heme-requiring cytochrome C production is impaired
along with operation of the cytochrome C respiratory chain in the brain when neonate rats are
exposed to lead.
12.3.6.6 Summary of Effects of Lead on Vitamin D Metabolism There has recently been a grow-
ing awareness of the interactions of lead and the vitamin D-endocrine system. A recent study
has found that children with blood lead levels of 33-120 ug/dl showed significant reductions
in serum levels of the hormonal metabolite 1,25-dihydroxyvitamin D (1,25-(OH)2D). This
inverse dose-response relationship was found throughout the range of measured blood lead
values, 12-120 ug/dl, and appeared to be the result of lead's effect on the production of the
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vitamin D hormone. The 1,25-(OH)2D levels of children with blood lead levels of 33-55 (jg/dl
corresponded to the levels that have been observed in children with severe renal dysfunction.
At higher blood lead levels (>62 ug/dl), the 1,25-(OH)2D values were similar to those that
have been measured in children with various inborn metabolic disorders. Chelation therapy of
the lead-poisoned children (blood lead levels >62 ug/dl) resulted in a return to normal
1,25-(OH)2D levels within a short period.
In addition to its well-known actions on bone remodeling and intestinal absorption of
minerals, the vitamin D hormone has several other physiological actions at the cellular level.
These include cellular calcium homeostasis in virtually all mammalian cells and associated
calcium-mediated processes that are essential for cellular integrity and function. In addi-
tion, the vitamin D hormone has newly recognized functions that involve cell differentiation,
immunoregulatory capacity, and other roles distinct from mineral metabolism. It is reasonable
to conclude, therefore, that impaired production of 1,25-(OH)2D can have profound and
pervasive effects on tissues and cells of diverse type and function throughout the body.
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12.4 NEUROTOXIC EFFECTS OF LEAD
12.4.1 Introduction
^Historically, neurotoxic effects have long been recognized as being among the more severe
consequences of human lead exposure (Tanquerel des Planches, 1839; Stewart, 1895; Prendergast,
1910; Oliver, 1911; Blackfan, 1917). Since the early 1900s, extensive research has focused on
the elucidation of lead exposure levels associated with the induction of various types of neu-
rotoxic effects and related issues, such as critical exposure periods for their induction and
their persistence or reversibility. Such research, spanning more than 50 years, has provided
increasing evidence indicating that progressively lower lead exposure levels, previously ac-
cepted as "safe," are actually sufficient to cause notable neurotoxic effects.
The neurotoxic effects of extremely high exposures, resulting in blood lead levels in ex-
cess of 80-100 ug/dl, have been well documented, especially in regard to increased risk for
fulminant lead encephalopathy (a well-known clinical syndrome characterized by overt symptoms
such as gross ataxia, persistent vomiting, lethargy, stupor, convulsions, and coma). The per-
sistence of neurological sequelae in cases of non-fatal lead encephalopathy has also been well
established. The neurotoxic effects of subencephalopathic lead exposures in both human adults
and children, however, continue to represent a major area of interest and controversy. Re-
flecting this, much research during the past 10-15 years has focused on the delineation of
exposure-effect relationships for the following: (1) the occurrence of overt signs and symp-
toms of neurotoxicity in relation to other indicators of subencephalopathic overt lead intoxi-
cation; and (2) the manifestation of more subtle, often difficult-to-detect indications of
altered neurological functions in apparently asymptomatic (i.e., not overtly lead-poisoned)
individuals.
The present assessment critically reviews the available scientific literature on the neu-
rotoxic effects of lead, first evaluating the results of human studies bearing on the subject
and then examining pertinent animal toxicology studies. The discussion of human studies is
divided into two major subsections focusing on neurotoxic effects of lead exposure in (1)
adults and (2) children. Lead's effects on both the central nervous system (CNS) and the
peripheral nervous system (PNS) are discussed in each case. In general, only relatively brief
overview summaries are provided in regard to findings bearing on the effects of extremely
high-level exposures resulting in encephalopathy or other frank signs or symptoms of overt
lead intoxication. Studies concerning the effects of lower-level lead exposures are assessed
in more detail, especially those dealing with non-overtly lead intoxicated children. As for
the animal toxicology studies, particular emphasis is placed on the review of studies that
help to address certain important issues raised by the human research findings, rather than
attempting an exhaustive review of all animal toxicology studies concerning the neurotoxic
effects of lead.
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12.4.2 Human Studies
Defining exposure-effect or dose-response relationships between lead and particular
neurotoxic responses in humans involves two basic steps. First, there must be an assessment
of the internal lead burden resulting from external doses of lead received via various routes
of exposure (such as air, water, food, occupational hazards, house dust, etc.). Internal lead
burdens may be indexed by lead concentrations in blood, teeth, or other tissue, or by other
biological indicators. The second step involves an assessment of the relationship of internal
exposure indices to behavioral or other types of neurophysiological responses. The difficulty
of this task is reflected by current controversies over existing data. Studies vary greatly
in the quality of design, precision of assessment instruments, care in data collection, and
appropriateness of statistical analyses employed. Many of these methodological problems are
broadly common to research on toxic agents in general and not just to lead alone.
Although epidemiological studies of lead's effects have immediate environmental relevance
at the human level, difficult problems are often associated with the interpretation of the
findings, as noted in several reviews (Bornschein et al. , 1980; Cowan and Leviton, 1980;
Rutter, 1980; Valciukas and Lilis, 1980; Needleman and Landrigan, 1981). The main problems
are the following: (1) inadequate markers of exposure to lead; (2) insensitive measures of
performance; (3) bias in selection of subjects; (4) inadequate handling of confounding co-
variates; (5) inappropriate statistical analyses; (6) inappropriate generalization and inter-
pretation of results; and (7) the need for "blind" evaluations by experimenters and techni-
cians. Each of these problems is briefly discussed below.
Each major exposure route—food, water, air, dust, and soil—contributes to a person's
• total daily intake of lead (see Chapters 7 and 11). The relative contribution of each expo-
sure route, however, is difficult to ascertain; neurotoxic endpoint measurements, therefore,
are most typically evaluated in relation to one or another indicator of overall internal lead
body burden. Subjects in epidemiological studies may be misclassified as to exposure level
unless careful choices of exposure indices are made based upon the hypotheses to be tested,
the accuracy and precision of the biological media assays, and the collection and assay pro-
cedures employed. Chapter 9 of this document evaluates different measures of internal expo-
sure to lead and their respective advantages and disadvantages. The most commonly used mea-
sure of internal dose is blood lead concentration, which varies as a function of age, sex,
race, geographic location, and exposure. The blood lead level is a useful marker of current
exposure but generally does not reflect cumulative body lead burdens as well as lead levels in
teeth. Hair lead levels, measured in some human studies, are not viewed as reliable indica-
tors of internal body burdens at this time. Future research may identify a more standard
exposure index, but it appears that a risk classification similar to that of the U.S. Centers
for Disease Control (1978) in terms of blood lead and free erythrocyte protoporphyrin (FEP)
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levels will continue in the foreseeable future to be the standard approach most often used for
lead exposure screening and evaluation. Much of the discussion below is, therefore, focused
on defining dose-effect relationships for human neurotoxic effects in terms of blood lead
levels; some ancillary information on pertinent tooth lead levels is also discussed.
The frequency and timing of sampling for internal lead burdens represent another impor-
tant factor in evaluating studies of lead effects on neurological and behavioral functions.
For example, epidemiological studies often rely on blood lead and/or erythrocyte protoporphy-
rin (EP) levels determined at a single point in time to retrospectively estimate or character-
ize internal exposure histories of study populations that may have been exposed in the past to
higher levels of lead than those indicated by a single current blood sample. Relatively few
prospective studies exist that provide highly reliable estimates of critical lead exposure
levels associated with observed neurotoxic effects in human adults or children, especially in
regard to the effects of subencephalopathic lead exposures. Some prospective longitudinal
studies on the effects of lead on early development of infants and young children are cur-
rently in progress, but results of these studies are only beginning to become available
(see Section 12.4.2.2.2.5 below). The present assessment of the neurotoxic effects of
lead in humans must, therefore, rely most heavily on published epidemiological studies which
typically provide exposure history information of only limited value in defining exposure-
effect relationships and less-than-optimum cross-sectional study designs.
Key variables that have emerged in determining effects of lead on the nervous system in-
clude (1) duration and intensity of exposure and (2) age at exposure. Much evidence suggests
that young organisms with developing nervous systems are more vulnerable than adults with
fully matured nervous systems. Particular attention is, therefore, accorded below to discus-
sion of neurotoxic effects of lead in children as a special group at risk.
Precision of measurement is a critical methodological issue, especially when research on
neurotoxicity leaves the laboratory setting. Neurotoxicity is often measured indirectly with
psychometric or neurometric techniques in epidemiological studies (Valciukas and Lilis, 1980).
The accuracy with which these tests reflect what they purport to measure (validity) and the
degree to which they are reproducible (reliability) are issues central to the science of mea-
surement theory. Many cross-sectional population studies make use of instruments that are
only brief samples of behavior thought to be representative of some relatively constant under-
lying traits, such as intelligence. Standardization of tests is the subject of much research
in psychometrics. The quality and precision of specific test batteries have been particularly
controversial issues in evaluating possible effect levels for neurotoxic effects of lead expo-
sure in children. Table 12A (Appendix 12A) lists some of the major tests used, together with
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their advantages and weaknesses. The following review places most weight on results obtained
with age-normed, standardized psychometric test instruments, and well-controlled, standardized
nerve conduction velocity tests. Other measures, such as reaction time, finger tapping, and
certain electrophysiological measures (e.g., cortical evoked and slow-wave potentials) are
potentially more sensitive indices, but are still experimental measures whose clinical utility
and psychometric properties with respect to the neurobehavioral toxicity of lead remain to be
more fully explored.
Selection bias is a critical issue in epidemiological studies in which attempts are made
to generalize from a small sample to a large population. Volunteering to participate in a
study and attendance at special clinics or schools are common forms of selection bias that
often limit how far the results of such studies can be generalized. These factors may need to
be balanced in lead neurotoxicity research since reference groups are often difficult to find
because of the pervasiveness of lead in the environment and the many non-lead covariates that
also affect performance. Selection bias and the effects of confounding can be reduced by
choosing a more homogeneous stratified sample, but the generalizability of the results of such
cohort studies is thereby limited.
Perhaps the greatest methodological concern in epidemiological studies is controlling for
confounding covariates, so that residual effects can be more confidently attributed to lead.
Among adults, the most important covariates are age, sex, race, educational level, exposure
history, alcohol intake, total food intake, dietary calcium and iron intake, and urban versus
rural styles of living (Valciukas and Lilis, 1980). Among children, a number of developmental
covariates are additionally important: parental socioeconomic status (Needleman etal.,
1979); maternal IQ (Perino and Ernhart, 1974); pica (Barltrop, 1966); quality of the care-
giving environment (Hunt et al., 1982; Milar et al., 1980); dietary iron and calcium intake;
vitamin D levels; body fat and nutrition (Mahaffey and Michaelson, 1980; Mahaffey, 1981); and
age at exposure. Preschool children below the age of 3-5 years appear to be particularly vul-
nerable, in that the rate of accumulation of even a low body lead burden is higher for them
than for adults (National Academy of Sciences, Committee on Lead in the Human Environment,
1980). Potential confounding effects of covariates become particularly important when trying
to interpret threshold effects of lead exposure. Each covariate alone may not be significant,
but, when combined, may interact to pose a cumulative risk which could result in under- or
overestimation of a small effect of lead.
Statistical considerations important not only to lead but to all epidemiological studies
include adequate sample size (Hill, 1966), the use of multiple regression (Cohen and Cohen,
1975), and the use of multivariate analyses (Cooley and Lohnes, 1971). Regarding sample size,
false negative conclusions are at times drawn from small studies with low statistical power.
It is often difficult and expensive to use large sample sizes in complex research such as that
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on lead neurotoxicity. This fact makes it all the more important to use sensitive assessment
instruments which have a high level of discriminating power and can be combined into factors
for multivariate analysis. Multiple statistical comparisons can then be made while reducing
the likelihood of finding a certain number of significant differences by chance alone. This
is a serious problem, because near-threshold effects are often small and variable.
A final crucial issue in this and other research revolves around the care taken to assure
that investigators are isolated from information that might identify subjects in terms of
their lead exposure levels at the time of assessment and data recording. Unconscious biases,
nonrandom errors, and arbitrary data correction and exclusion can be ruled out only if a study
is performed under blind conditions or, preferably, double-blind conditions.
With the above methodological considerations in mind, the following sections evaluate
pertinent human studies. The discussion includes an overview of lead exposure effects in
adults, followed by a more detailed assessment of neurotoxic effects of lead exposures in
children.
12.4.2.1 Neurotoxic Effects of Lead Exposure in Adults.
12.4.2.1.1 Overt lead intoxication in adults. Severe neurotoxic effects of extreme exposures
to high levels of lead, especially for prolonged periods that produce overt signs of acute
lead intoxication, are well documented in regard to both adults and children. The most pro-
found (CNS) effects in adults have been referred to for many years as the clinical syndrome of
lead encephalopathy, described in detail by Aub et al. (1926), Cantarow and Trumper (1944),
Cumings (1959), and Teisinger and Styblova (1961). Early features of the syndrome that may
develop within weeks of initial exposure include dullness, restlessness, irritability, poor
attention span, headaches, muscular tremor, hallucinations, and loss of memory. These symp-
toms may progress to delirium, mania, convulsions, paralysis, coma, and death. The onset of
such symptoms can often be quite abrupt, with convulsions, coma, and even death occurring very
rapidly in patients who shortly before appeared to exhibit much less severe or no symptoms of
acute lead intoxication (Cumings, 1959; Smith et al., 1938). Symptoms of lead encephalopathy
indicative of severe CNS damage and posing a threat to life are generally not seen in adults
except at blood lead levels well in excess of 120 ug/dl (Kehoe, 1961a,b,c). Other data (Smith
et al., 1938) suggest that acute lead intoxication, including severe gastrointestinal symptoms
and/or signs of encephalopathy can occur in some adults at blood lead levels around 100 ug/dl
but ambiguities make the data difficult to interpret.
In addition to the above CNS effects, lead also clearly damages peripheral nerves at tox-
ic, high-exposure levels that predominantly affect large myelinated nerve fibers (Vasilescu,
1973; Feldman et al., 1977; Englert, 1980). Pathologic changes in peripheral nerves, as shown
in animal studies, can include both segmental demyelination and, in some fibers, axonal degen-
eration (Fullerton, 1966). The former types of changes appear to reflect lead's effects on
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Schwann cells, with concomitant endoneurial edema and disruption of myelin membranes
(VMndebank and Dyck, 1981). Apparently, lead induces a breakdown in the blood-nerve barrier
which allows lead-rich edema fluid to enter the endoneurium (Dyck etal., 1980; Windebank
etal., 1980). Remye 11 nation observed in animal studies suggests either that such lead
effects may be reversible or that not all Schwann cells are affected equally (Lampert and
Schochet, 1968; Ohnishi and Dyck, 1981). Reports of plantar arch deformities due to old per-
ipheral neuropathies (Emmerson, 1968), however, suggest that lead-induced neuropathies of
sufficient severity in human adults could result in permanent peripheral nerve damage. Mor-
phologically, peripheral neuropathies are usually detectable only after prolonged high expo-
sure to lead, with distinctly different sensitivities and histological differences existing
among mammalian species. In regard to man, as an example, Buchthal and Behse (1979, 1981),
using nerve biopsies from a worker with frank lead neuropathy (blood lead = 150 ug/dl), found
histological changes indicative of axonal degeneration in association with reductions in nerve
conduction velocities that corresponded to loss of large fibers and decreased amplitude of
sensory potentials.
Data from numerous studies provide a basis by which to estimate lead exposure levels at
which adults exhibit overt signs or symptoms of neurotoxicity and to compare such levels with
those associated with other types of signs and symptoms indicative of overt lead intoxication
(Sakurai et al., 1974; Lilis etal., 1977; Tola and Nordman, 1977; Irwig etal., 1978a,b;
Dahlgren, 1978; Baker etal., 1979; Haenninen etal., 1979; Spivey etal., 1979; Fischbein
et al., 1980; Hammond et al., 1980; Kirkby et al., 1983). These studies evaluated rates of
various clinical signs and symptoms of lead intoxication across a wide range of lead exposures
among occupationally exposed smelter and battery plant workers.
Considerable individual biological variability is apparent among various study popula-
tions and individual workers in terms of observed lead levels associated with overt signs and
symptoms of lead intoxication, based on comparisons of exposure-effect and dose-response data
from the available studies. For example, Irwig et al. (1978a,b) and Zielhuis and Wibowo
(1976) discuss data for black South African lead workers indicative of increased prevalence of
neurological symptoms at 110 ug/dl and gastrointestinal symptoms at blood lead levels in ex-
cess of 60 ug/dl. Analogously, Hammond et al. (1980) reported significant increases in neuro-
logical (both CNS and PNS) and gastrointestinal symptoms among American smelter workers with
blood lead levels often exceeding 80 ug/dl, but not among workers whose exposure histories did
not include levels above 80 ug/dl. Also, Kirkby et al. (1983) found no significant differ-
ences between 96 long-term lead smelter workers and 96 matched control subjects in prevalence
of self-reported symptoms of fatigue, headache, nervousness, sleep disturbance, constipation,
or colic. Blood lead levels for the lead workers averaged 51 ug/dl (range 13-91 ug/dl),
whereas the control group averaged 11 ug/dl (range 6-16 ug/dl).
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In contrast to the above results, many other investigators have reported neurologic
symptoms and other overt signs and symptoms of lead toxicity at blood lead levels ranging well
below 80 jjg/dl. Lilis et al. (1977), for instance, found that CNS symptoms (tiredness,
sleeplessness, irritability, headaches) were reported by 55 percent and muscle or joint pain
by 39 percent of a group of lead smelter workers whose blood lead levels had never been ob-
served to exceed 80 ug/dl. Low hemoglobin levels (<14 g/dl) were found in more than 33
percent of these workers. In addition, Spivey et al. (1979) reported significantly increased
neurological (mainly CNS, but some PNS) symptoms and joint pain among a group of 69 lead
workers with mean ± standard deviation blood lead levels of 61.3 ± 12.8 ug/dl in comparison to
a control group with 22.0 ±5.9 ug/dl blood lead values. Haenninen et al. (1979) similarly
reported significantly increased neurological (both CNS and PNS) and gastrointestinal symptoms
among 25 lead workers with maximum observed blood lead levels of 50-69 ug/dl and significantly
increased CNS symptoms among 20 lower exposure workers with maximum blood lead values below
50 ug/dl. Both groups were compared against a referent control group (N = 23) with blood lead
values of 11.9 ±4.3 ug/dl.
Additional studies (Baker et al., 1979; Fischbein et al., 1980; Zimmermann-Tansella et
al., 1983) provide evidence of overt signs or symptoms of neurotoxicity occurring at lead
exposure levels still lower than those indicated above. Baker et al. (1979) studied dose-
response relationships between clinical signs and symptoms of lead intoxication among lead
workers in two smelters. No overt toxicity was observed at blood lead levels below 40 ug/dl.
However, 13 percent of those workers with blood lead values in the range 40-79 ug/dl had
extensor muscle weakness or gastrointestinal symptoms; and anemia occurred in 5 percent of the
workers with 40-59 ug/dl blood lead levels, in 14 percent with levels of 60-79 ug/dl, and in
36 percent with blood lead levels exceeding 80 ug/dl. Also, Fischbein et al. (1980), in a
study of 90 cable splicers intermittently exposed to lead, found higher zinc protoporphyrin
levels (an indicator of impaired heme synthesis associated with lead exposure) among workers
reporting CNS or gastrointestinal symptoms than among other cable splicers not reporting such
symptoms. Only 5 percent of these workers had blood lead levels in excess of 40 ug/dl, and
the mean ± standard deviation blood lead levels for the 26 reporting CNS symptoms were 28.4
±7.6 ug/dl and 30 ± 9.4 ug/dl for the 19 reporting gastrointestinal symptoms. However,
caution must be exercised in accepting these latter blood levels as being representative of
average or maximum lead exposures of this worker population, in view of the highly intermit-
tent nature of their exposure and the likelihood of much higher peaks in their blood lead
levels than those coincidentally measured at the time of their blood sampling.
Lastly, Zimmermann-Tansella et al. (1983) have independently confirmed and extended pre-
viously described findings of Haenninen et al. (1979). Three groups of 20 men each were
matched on age, education, marital status, chronic illnesses, personality characteristics, and
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length of employment. The control group had no history of occupational exposure to lead (mean
blood lead level = 20.4 ± 6 H9/dl). The lead-exposed groups were composed of workers from an
electric storage battery plant, with the low-lead group averaging 31.7 ±2.9 ug/dl (range:
26-35 Mg/dl) and tne high-lead group 52.5 ± 5.1 ug/dl (range: 45-60 ug/dl). None ever ex-
ceeded 60 ug/dl. In conjunction with other psychological testing (Campara et al., 1984), two
questionnaires were given that asked about a variety of emotional, neurological, and gastro-
intestinal symptoms similar to symptoms covered by the questionnaire used by Haenninen et al.
(1979). The most clear-cut effects, in terms of significant and consistent dose-response
trends, were found in physical symptoms (such as loss of appetite, paresthesis in lower limbs,
weakness of upper limbs, and dropping of objects) with the most marked increases seen in rates
of neurological symptoms in the high-lead group. Coupled with the increased symptom rates
observed by Zimmermann-Tansella et al. (1983) were observations reported by Campara et al.
(1984) indicating that the high-lead workers did significantly more poorly on a variety of
psychometric tests (e.g., the WAIS), with general performance (on cognitive and visual-motor
coordination tasks) and verbal reasoning ability most markedly impaired. These findings,
consistent with earlier results of Haenninen et al. (1978, 1979), indicate that overt neurol-
ogical symptoms and impaired CMS functioning, as well as gastrointestinal symptoms, occur in
adults at blood lead levels of 45-60 ug/dl.
Overall, the results reviewed above appear to support the following conclusions: (1)
overt signs and symptoms of neurotoxicity in adults are manifested at roughly comparable lead
exposure levels as other types of overt signs and symptoms of lead intoxication, such as gas-
trointestinal complaints; (2) neurological signs and symptoms are indicative of both central
and peripheral nervous system effects; (3) such overt signs and symptoms, both neurological
and otherwise, occur at markedly lower blood lead levels than levels previously thought to be
"safe" for adults; and (4) lowest observed effect levels for the neurological signs and symp-
toms in adults can most credibly be stated to be in the 40-60 ug/dl range. Insufficient in-
formation currently exists to estimate with confidence to what extent or for how long such
overt signs and symptoms persist in adults after termination of precipitating external lead
exposures, but at least one study (Dahlgren, 1978) has reported abdominal pain persisting as
long as 29 months after exposure termination among 15 smelter workers, including four whose
blood lead levels were between 40 and 60 ug/dl while working.
12.4.2.1.2 Non-overt lead intoxication in adults. Of special importance for establishing
standards for exposure to lead is the question of whether exposures lower than those producing
overt signs or symptoms of lead intoxication result in less obvious neurotoxic effects in
otherwise apparently healthy individuals. Attention has focused in particular on whether ex-
posures leading to blood lead levels below 80-100 ug/dl may lead to behavioral deficits or
other neurotoxic effects in the absence of classical signs of overt lead intoxication.
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In adults, one might expect neurobehavioral deficits to be reflected by performance mea-
sures in the workplace, such as higher rates of absences or reduced psychomotor performance
among occupationally exposed lead workers. Some epidemiological studies have investigated
possible relationships between elevated blood lead and general health as indexed by records of
sick absences certified by physicians (Araki et al., 1982; Robinson, 1976; Shannon et al.,
1976; Tola and Nordman, 1977). However, sickness absence rates are generally poor epidemio-
logic outcome measures that may be confounded by many variables and are difficult to relate
specifically to lead exposure levels. Much more useful are studies that evaluate direct
measurements of central or peripheral neurological functions in relation to lead exposure.
A number of studies have employed sensitive neurological and/or psychometric testing pro-
cedures in an effort to demonstrate specific lead-induced neurobehavioral effects in adults.
Disturbances in oculomotor function have been found in two studies of lead-exposed workers.
The first, a prospective investigation by Baloh et al. (1979), found significantly decreased
saccade accuracy and similar but nonsignificant differences in saccade velocity and delay
times in lead workers (mean blood lead: ~61 ug/dl) compared to controls. A follow-up examina-
tion (Spivey et al., 1980) essentially replicated the original findings. A more recent
investigation of saccadic eye movements by Glickman et al. (1984) also found highly signifi-
cant decreases in saccade accuracy and increases in overshoots among lead workers (mean blood
lead: 57 ug/dl), particularly younger workers. The difference in saccade velocity fell just
short of statistical significance overall (p = 0.056), but was highly significant (p <0.004)
in the 20-29 year age group. Also, velocity and ZPP were significantly correlated overall
(r = -0.40, p <0.005).
Morgan and Repko (1974) reported deficits in hand-eye coordination and reaction time in
an extensive study of behavioral functions in 190 lead-exposed workers (mean blood lead level
= 60.5 ± 17.0 ug/dl). The majority of the subjects had been exposed between 5 and 20 years.
In a similar study, however, Milburn et al. (1976) found no differences between control and
lead-exposed workers on numerous psychometric and other performance tests. On the other hand,
several other studies (Arnvig et al., 1980; Grandjean et al., 1978; Haenninen et al., 1978;
Hogstedt et al., 1983; Mantere et al., 1982; Valciukas et al., 1978) have found disturbances
in reaction time, visual motor performance, hand dexterity, IQ test/cognitive performance,
mood, nervousness, or coping in lead workers with blood lead levels of 50-80 ug/dl. Hogstedt
et al. (1983) also found impaired memory and learning ability in workers with time-weighted
average blood lead levels of 27-52 ug/dl. Furthermore, Baker et al. (1983) found significant-
ly increased rates of depression, confusion, anger, fatigue, and tension among workers with
blood lead levels above 40 ug/dl, who did not differ from referent control workers in terms of
reported incidence of abdominal colic or other gastrointestinal symptoms characteristic of
overt lead intoxication. Other aspects of neurobehavioral function in the same workers were
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also found to be impaired, including verbal concept formation, memory, and visual/motor per-
formance. A graded dose-effect relationship for non-overt CMS lead effects in otherwise
asymptomatic adults is indicated by such studies.
In addition to the above studies indicative of psychoneural dysfunctions in non-overtly
lead intoxicated adults, numerous investigations have examined peripheral nerve function by
measuring the conduction velocity of electrically stimulated nerves in the arm or leg. Nerve
conduction velocity (NCV) provides a readily accessible indication of neurophysiological
function in sensory as well as motor nerves. However, nerve temperature (and to some extent,
skin temperature and room temperature), age, and limb length affect conduction velocity and
thus may confound NCV measurements. Table 12-1 summarizes several studies of groups of
lead-exposed subjects and notes what was done to deal with the above confounding factors.
Of particular note are the positive findings of decreased NCVs at blood lead levels of
30-50 |jg/dl from a prospective occupational study by Seppalainen et al. (1983) in contrast to
the negative findings at blood lead levels of 60-80 M9/dl from a prospective study by Spivey
et al. (1980). Also contrasting are the results of two cross-sectional studies: Rosen et al.
(1983) observed significant slowing of NCVs as a function of average blood lead levels moni-
tored over 9 years, whereas Triebig et al. (1984) reported no apparent dose-effect relation-
ship on NCVs except at blood lead levels exceeding 70 ug/dl. Although Triebig et al. (1984)
did not examine as many neurophysiological variables as Rosen et al. (1983), they did incor-
porate many more lead-exposed subjects (N = 133) compared to most occupational lead studies of
NCV. Triebig et al. (1984) also noted that the earlier findings of Seppalainen et al. (1975,
1979) were confounded by age effects, since their lead-exposed subjects were older than the
controls, but no correction was made for the normal slowing of NCV with increasing age.
However, even if one allows -a decline of approximately 2 m/sec in NCV for each 10 years' in-
crease in age (based on Triebig et al., 1984), the age differences in Seppalainen et al.
(1983) would not appear to be sufficient to account for the significant declines in NCV that
they found, except possibly at the four-year stage of their longitudinal study. Moreover,
Rosen et al. (1983) did include age as a covariate in their analyses and still found signi-
ficant effects of lead on NCV and other neurophysiological variables.
One difficulty in drawing conclusions from the studies presented in Table 12-1 is the
lack of consistency among studies, either in the nerves examined or in the significance of re-
sults obtained. No one nerve has been consistently used in all the studies dealing with lead
exposure and NCV measurements. Even when a particular nerve is singled out for consideration,
the results may not be in complete agreement. For example, Seppalainen et al. (1975, 1979)
initially found the ulnar slow fiber NCVs to be a sensitive indicator of lead-induced impair-
ment. But more recent work by Seppalainen et al. (1983) and some other investigators (e.g.,
12-61
-------
TABLE 12-1. SUMMARY OF STUDIES ON NERVE CONDUCTION VELOCITY IN GROUPS OF LEAD-EXPOSED SUBJECTS
CTi
ro
Mean blood lead,
ug/dl
Reference
Seppatainen
et al. (1983)
Corsi et al.
(1984)
Johnson et al.
(1980)
Seppala'inen
et al. (1979)
Exp. Con.
(range/±S.D.)
16
(7-30)
31
(13-48)
30
(13-48)
27
(17-37)
28
(•ax: 71-
180 4 yr
or more
earlier)
9 30(±14)
* 56(113)
34 actual
45 THAT
(•ax: <70)
10
(1-21)
10
(7-18)
10
(5-21)
7
(4-12)
20
10(±4)
15(±6)
10
Exposure No. of
period, yr. subjects
(range/lS. D. ) Exp. Con.
0 23 23
1 23 23
2 15 15
4 10 10
7 38 23
(0.08-37)
(3-27 yr
since last
exposure)
2.4(±1.5) 45 31
12.4(±10) 164 108
~8 61 34
NCV,
m/sec %
Nerve*
Med
Med
Uln
Uln
Med
Hed
Uln
Uln
Hed
Hed
Uln
Uln
Hed
Hed
Uln
Uln
Uln
\i\n
Per
Per
Uln
Per
Uln
Per
Hed
Hed
Uln
Uln
Uln
Per
Tib
Sur
- n
- s
- •
- s
- •
- s
- •
- s
- n
- s
- m
- s
- n
- s
- M
- S
- m
- sf
- m
- sf
- a
- m
- m
- m
- m
- s
- n
- s
- sf
- n
- m
- s
Exp.
58.8
63.7
60.7
62,1
55.7
60.5
57.0
59.2
54.8
58.5
59.3
59.9
59.1
59.9
62.8
63.2
54.5
49.9
48.7
45.2
58. 8
52.3
56.5
49.4
59.6
62.0
60.7
60.9
42.5
54.6
48.6
43.9
Con.
60.7
64.2
59.3
62.7
61.5
64.2
61.5
64.6
60.4
60.7
62.2
60.8
64.5
61.1
62.9
66.1
57.9
54.0
51.3
48.5
61.5
55.2
57.1
50.4
61.0
65.3
60.5
63.1
45.9
54.3
50.7
43.9
Diff.
-3
-1
+2
-1
-9
-6
-7
-8
-9
-4
-5
-2
-8
-2
0
-4
-6
-8
-5
-7
-4
-5
-1
-2
-2
-5
0
-4
-7
+1
-4
0
P
N.S.
N.S.
N.S.
N.S.
<0.01
<0.05
<0.01
<0.01
-------
TABLE 12-1. (continued)
ro
i
Mean blood lead,
ug/dl
Reference
Seppalainen
et al. (1975)
Verberk (1976)
Exp.
(range/±S
40
Con.
.0.)
10-13
(28-65) (estimated)
40
20
Exposure
period, yr.
(range/±S.D. )
4.6
(1-17)
Overal 1
49 days
No
. of
NCV,
subjects
Exp.
26
25
26
26
25
25
26
= 26
10
Con.
26
8
26
22
23
26
19
= 26
9
Nerve*
Hed - m
Hed - s
Uln - m
Uln - sf
Uln - s
Per - m
Tib - m
Uln - n
Uln - sf
IT/ sec %
Exp.
54.5
59.5
55.0
42.0
58.2
50.6
43.4
57.4
46.2
Con.
58.5
56.3
58.1
47.1
60.0
52.0
44.6
59.2
50.8
Diff.
-7
+6
-5
-11
-3
-3
-3
-3
-9
P
<0.005
N.S.
<0.01
<0.001
N.S.
N.S.
N.S.
N.S.
N.S.
Comments
Con. group values ob-
tained from separate
studies.
Volunteers ingested Pb. No
info, on skin temperature
Rosen et al.
(1983)
Bordo et al.
(19B2a,b)
Araki & Honma
(1976)
>40 <25
(«ax: >55) (max: 30)
(0.5-28)
42(112)
(avg. <50
during preced-
ing 24 no.)
45
(29-73)
16(t3)
12
4
(O.b-10)
18
(0.67-46)
15
16
(intermediate
PbB
N
62
group
= 8)
27
Uln
Hed
Fib
Tib
Uln
Hed
Sur
Hed
Hed
Per
- m
- m
- m
- m
- sf
- s
- s
- m
- s
- m
57
55
45.
44
40.
48.
43.
59.
59.
51.
.0
.8
.7
.0
7
7
1
7
8
8
58.
56.
48.
45.
43.
48.
49.
63.
63.
51.
1
9
1
7
2
7
1
1
5
4
-2
-2
-5
-4
-6
0
-12
-5
-6
+1
N
N
0,
N.
N.
N.
D.
<0.
<0.
N.
.5.
.5.
,019
S.
S.
S.
022
01
01
S.
19
39
Hed - m
Hed - nx
Tib - m
54.3
64.1
44.7
59.0
67.1
50.0
-8
-4
-11
<0.01
N.S.
<0.01
or age comparisons. Resi-
dual error > difference be-
tween groups; ulnar-sf NCVs
12-13X faster in both
groups compared to pre-
exposure values.
NCV values adjusted for
age. Cumulative exposure
showed no apparent
effect.
ANACOVA included age as
covariate. Duration of
expos, showed no effect.
Room temperature mea-
sured, but not skin
temperature. No info.
on age of Con. Ss.
-------
TABLE 12-1. (continued)
ro
en
Mean blood lead,
u9 89
62
86
Overall = 95
? 14
Con.
20
20
18
16
50
20
20
19
19
20
43
44
29
29
28
58
64
21
14
21
= 21
N/A
Nerve*
Uln
Uln
Rad
Rad
Uln
Uln
Rad
Rad
Med
Uln
Uln
Uln
Tib
Med
Uln
Uln
Med
Med
Sur
Tib
Med
Rad
Per
Med
- m
- sf
- m
- sf
- m
- sf
- m
- sf
- m
- m
- s
- sf
- m
- m
- m
- sf
- m
- s
- s
- m
- m
- m
- m
- m
NCV,
m/sec %
Exp.
55.2
48.5
62.1
41.7
52.4
45.1
65.2
51.3
53.4
55.6
56.4
48.0
50.5
55.0
55.2
34.3
54.7
46.7
44.2
47.2
56
67
48
52
Con.
55.7
49.4
65.2
51.2
53.6
48.7
68.0
49.7
59.9
64.5
63.0
45.7
55.5
55.1
56.1
33.2
61.3
45.7
44.9
49.6
56
64
48
58
om.
-1
-2
-5
-19
-2
-7
-4
+3
-11
-14
-10
+5
-9
0
-2
+3
-11
+2
-2
-5
0
+5
0
-11
P
N.S.
N.S.
N.S.
N.S.
N.S.
<0.05
N.S.
N.S.
0.00003
0.00003
0.015
N.S.
0.0013
N.S.
N.S.
N.S.
<0.0001
N.S.
N.S.
<0.025
N.S.
N.S.
N.S.
<0.01
Comments
Ulnar NCV values corrected
for skin temperature. Male
groups age-matched, female
Exp. group 3 yr older than
Con. group. Although com-
bined radial-sf values not
reported as statistically
significant, difference be-
tween female groups for
radial-sf NCVs appears to
be significant despite high
variance.
Skin temperature controlled
and corrected for data
analysis. Only sig. corre-
lation between PbB and NCV
was for ulnar-m.
Exp. group ~3 yr older than
Con. group. No info, on
skin temperature.
Con. values obtained from
separate independent study.
Room temperature and skin
impedance controlled, but
not skin temp.
Mean age of Exp. and
Con. groups equal, but
no info, on subsets of
Ss used in different
NCV tests.
Exp. Ss treated with EOTA
to reduce PbB; no sepa-
rate Con. group. Difference
between Exp. and Con.
values reflects change over
1-mo to 3-yr period after
EDTA treatment.
-------
TABLE 12-1. (continued)
no
i
01
01
Reference
Paulev et al.
(1979)
Tri«big et al.
(1984)
Ashby (1980)
Singer et al.
(1983)
Spivey et al.
(1980)
Sborgia et al.
(1983)
Mean blood lead,
pg/dl Exposure No. of
Exp. Con. period, yr. subjects
(range/±S.D. ) (range/±S.O. ) Exp. Con.
53 11 12.9 mo 32 14
(±16) (±4) (2-37 mo)
53 actual <20 11 133 66
(22-90) (1-28)
54 TWA2
60 ? 0.5-33 94 94
58(±16) 24{±14) <2 13 13
-(man: <80)
60 ? 10.6 37 26
(±17) (0.5-28) 35 24
25 13
24 20
Overall = 4TJ 31
60 22 21 55 31
(±11.9) (±6.2)
66 28 1-1.5 yr later 55 31
(±12.5) (±8.4)
63 24 7.84 31 35
(i!9.0) (±7.4)
Nerve*
Uln -
Uln -
Uln -
Uln -
Uln -
Med -
Uln -
Med -
Rad -
Per -
Uln -
Uln -
Med -
Rad -
Per -
Med -
Hed -
Per -
Sur -
Uln -
Uln -
Per -
Uln -
Ulo -
Per -
Med -
Med -
Ked -
Med -
Uln -
Pop -
Tib -
n(lf>
m(rt)
m
s
s(d)
m
n
m
n
m
s
o
m
m
»
m
s
m
s
n
sf
m
in
sf
ra
m
sf
s(d)
s(p)
n
m
D
NCV,
m/sec %
Exp.
58.8
58.8
58.3
52.3
45.5
46.1
53.4
55.9
63.9
46.1
57.5
55.1
58.4
58.1
46.6
56.1
42.9
49.0
37.8
55.5
45.5
52.3
56.2
45.3
50. S
55.4
50.1
63.4
61.2
55.0
49.3
48.7
Con.
55.3
53.7
59. Z
53.1
47.5
47.1
55.6
57.3
71.7
47.6
57.9
58.0
59.8
74.1
49.9
57.6
46.8
49.2
42.8
56.0
46.9
51.5
53.1
44.1
48.9
56.6
49.7
62.7
61.0
54.5
50.0
46.4
Diff.
+6
+9
-2
+2
-4
-2
-4
-2
-11
-3
-1
-5
-2
-22
-7
-3
-8
0
-12
-1
-3
+2
+6
+3
+3
-2
+1
+1
0
+1
-1
+5
P
N.S.
N.S.
N.S.
N.S.
50.05
SO. 05
<0.0005
<0.01
<0. 0005
<0. 005
N.S.
<0.05
N.S.
<0.005
<0.05
0.36
0.006
0.87
.0.0004
M.S.
N.S.
N.S.
0.027
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
Comments
Room temperature con-
trolled; no info, on skin
temperature. Groups com-
parable in height, weight,
and age range.
Analysis of subgroup! ngs
of Exp. Ss by PbB indicated
that effects primarily
seen at £70 ug/dl.
Anomalous pos. correlation
observed between ulnar-ra
NCV and PbB; possibly
sig. due to use of multiple
t-tests. Skin temperature
of Con. Ss < Exp. Ss.
Separate analysis of
subset of Ss from main
study; linn ted to new
employees of <2'yr.
Adjustment of NCVs for
age and skin tenperature
increased statistical
significance of effects;
for Ss exposed >10 yr,
median-la NCV also sig-
nificantly slower.
Exp. group 2.8 yr
older than Con. group.
Sig. (<0.05) neg. corr.
between ulnar-n NCV
and age. Room and skin
temperature controlled.
NCVs corrected for skin
tenperature. Regression
analysis showed no sig.
effect of max. or past
avg. PbB, but cnax. ZPP
showed sig. association
with ulnar NCV.
-------
TABLE 12-1. (cDntinued)
cr,
cr.
Reference
Baker et al.
(1984)
Feldnan et al.
(1977)
Englert (1980)
Vasilescu (1973)
Melgaard et al.
(1976)
Catton et al.
(1970)
Buchthal A
Sense (1979)
Mean blood lead,
ua/dl
Exp. Con.
(range/±S.O. )
(60-80) (0-20)
70 ?
(ia-no)
71
(•ax: 140)
72 ?
(27-180)
78 -19
(38-125) (2-36)
(40-120+) ?
(70-14B) ?
(•ax.
during
preceding
year)
Exposure No. of
period, yr. subjects
(range/±S.D.) Exp. Con.
-2-3 5 20
(0-20) (intermediate
PbB groups
N = 92)
? 19 25
? 99 0
? 50 30
? 20 12
(0.4-13) 19 17
(0.33-33) 20 ?
Nerve*
Uln -
Uln -
Per -
Sur -
Per -
Uln -
Uln -
Med -
Uln -
Per -
Rad -
Per -
Uln -
Uln -
Pop -
Pop -
Med -
Med -
Per -
Sur -
m
s
m
s
m
m
sf
m
n
n
•
m
n
s
ID
S
„
S
m
s
NCV,
m/sec
Exp.
63.4
55.1
52.6
45.4
45.0
58.8
-47
55.6
56.6
46.1
44.8
48.7
55.0
-57.0
49.9
57.1
58.1
63.7
50.1
50.7
Con.
62.5
51.8
50.8
48.7
54.1
.
-
57.2
57.3
60.5
49.8
-50
-58
-54
49.6
56.3
63.6
67.1
51.0
54.6
%
Diff.
+1
+6
+4
-7
-17
_
-
-3
-1
-24
-10
-3
-5
+6
0
+1
-9
-5
-2
-7
P
N.S.
0.02
N.S.
0.03
<0.02
N.S.
N.S.
0.05
0.05
0.05
0.05
7
?
7
N.S.
N.S.
<0.001
<0.01
N.S.
<0.001
Contents
p-values refer to an
exposure coefficient
(based on 12-no TYft2)
in a multiple linear
regression Model allowing
for age, height, weight,
and skin temperature across
all PbB groups.
No info, on Con. Ss or on
ages of Exp. Ss. Skin
temperature not controlled.
Ho sig. correlation be-
tween NCV and PbB, but
sig. slowing at high ALA-U
levels. NCV corrected for
age, skin temperature,
and body height.
P values as reported. No
info, on skin temperature.
Exp. group about 2 yr
younger than Con. group.
Con. values estimated from
info, in report. MCVs
corrected for skin tempera-
ture but not age. Ss
exposed to other aetals
besides Pb.
Only sig. difference be-
tween groups was in ratio
of knee and ankle muscle
action potentials.
Ho histological evidence
of abnormality in sural
nerve. No info, on how
Con. values obtained,
but said to be matched
for age.
*Med = median; Uln = ulnar; Per = peroneal; Tib = posterior tibial; Sur = sural; Fib - fibular; Pop = lateral popliteal;
m = motor; s = sensory; nx = mixed; sf = slow fibers; If = left; rt = right; d = distal; p = proximal.
tTVA = tine-weighted average. *
-------
Rosen et al., 1983; Spivey et al., 1980) has failed to find significant effects with ulnar
slow fibers. Nevertheless, the preponderance of effects has been in the negative direction,
as reflected in the column of Table 12-1 showing the percent differences in NCVs between
lead-exposed and referent groups. Moreover, of the various nerves examined, the most con-
sistently decreased NCVs appear to involve the median motor nerve. Recent experimental work
with rats may help explain this finding. Bouldin et al. (1985) found that lead-treated rats
showed a greater susceptibility to demyelination in the sciatic nerve (a mixed nerve contain-
ing a large number of motor fibers) than in the sural nerve (a sensory nerve). Given the
dependence of nerve conduction on the functional integrity of the nerve's myelin sheath, this
difference in susceptibility would help explain the variability in results of NCV studies
examining different types of nerves and is consistent with the emergence of the median motor
nerve as the most prevalent indicator of reduced NCV in the studies listed in Table 12-1.
A problem inherent in nearly all NCV studies has been the lack of experimental manipula-
tion of the presumed cause of lowered conduction velocities. Of interest in this regard is
the study of Araki et al. (1980), in which median motor NCVs were measured before and after
blood lead levels were lowered through chelation therapy. The investigators found that, de-
pending on a worker's initial NCV and the amount of change in blood lead level achieved
through chelation, significantly improved nerve conduction was measured in 7 of 14 lead-
exposed workers. For all subjects considered together, the increase in NCV correlated sig-
nificantly with the decrease in blood lead level (r = -0.573, p <0.001). The work of Araki et
al. (1980), as well as case studies reported by Feldman et al. (1977), indicate that lead-
induced impairment of nerve conduction is reversible, at least in part, by a reduction in
blood lead levels through chelation therapy. Although helpful in establishing a causal con-
nection between lead exposure and peripheral nerve function, these studies have not resolved
the dispute over whether such effects merely reflect mild, fully reversible impacts of lead
(Buchthal and Behse, 1981) or are true early warning signals of progressively more serious
neuropathy in otherwise undiagnosed lead intoxication (Feldman et al., 1977; Seppalainen and
Hernberg, 1980).
Taken as a whole, the studies reviewed here indicate the likelihood of NCV effects at
blood lead levels below 70 ug/dl, possibly even as low as 30 ug/dl, although further prospec-
tive studies are needed to characterize these levels definitively. It is important to note
that even though many of the observed changes in NCV may fall within the range of normal
variation, these studies show significant effects in groups of subjects, not just individual
subjects. Thus, these effects clearly represent departures from normal neurological func-
tioning and should be seriously considered for their potential health significance.
12-67
-------
12.4.2.1.3 Other Hypothesized Neurotoxic Effects of Lead in Adults. There are several case
reports of previous overexposure to heavy metals, e.g. lead, in amyotrophic lateral sclerosis
(ALS) patients and patients dying of motor neuron disease (MND). These reports have led to
hypotheses concerning the relationship between such neurotoxic syndromes and lead exposure
Conradi et al. (1976, 1978a,b, 1980), for example, found elevated lead levels in the cerebro-
spinal fluid of ALS patients as compared with controls. In addition, Kurlander and Patten
(1979) found that lead levels in spinal cord anterior horn cells of MND patients were nearly
three times that of control subjects and that lead levels correlated with illness durations-
despite chelation therapy for about a year, high lead levels remained in their tissue. On the
other hand, certain other studies (e.g., Manton and Cook, 1979; Stober et al., 1983) have not
found evidence to support an association of lead exposure with ALS. Thus, the evidence for
possible pathogenic significance of lead in ALS and motor neuron disease is at best mixed at
this time and the issue needs to be further explored by future research.
12.4.2.2 Neurotoxic Effects of Lead Exposure in Children.
12.4.2.2.1 Overt lead intoxication in children. Symptoms of encephalopathy similar to those
that occur in adults have been reported to occur in infants and young children (Prendergast
1910; Oliver, 1911; Blackfan, 1917; McKhann and Vogt, 1926; Giannattasio et al., 1952-
Cumings, 1959; Tepper, 1963; Chisolm, 1968), with a markedly higher incidence of severe en-
cephalopathic symptoms and deaths occurring among them than in adults. This may reflect the
greater difficulty in recognizing early symptoms in young children, thereby allowing intoxica-
tion to proceed to a more severe level before treatment is initiated (Lin-Fu, 1973). jn
regard to the risk of death in children, the mortality rate for encephalopathy cases was ap-
proximately 65 percent prior to the introduction of chelation therapy as standard medical
practice (Greengard et al., 1965; National Academy of Sciences, 1972; Niklowitz and Mandybur
1975). The following mortality rates have been reported for children experiencing lead en-
cephalopathy since the inception of chelation therapy as the standard treatment approach: 39
percent (Ennis and Harrison, 1950); 20-30 percent (Agerty, 1952); 24 percent (Mellins and
Jenkins, 1955); 18 percent (Tanis, 1955); and 5 percent (Lewis et al., 1955). These data, and
those tabulated more recently (National Academy of Sciences, 1972), indicate that once lead
poisoning has progressed to the point of encephalopathy, a life-threatening situation clearly
exists and, even with medical intervention, is apt to result in a fatal outcome. Historically
there have been three stages of chelation therapy. Between 1946 and 1950, dimercaprol (BAL)
was used. From 1950 to 1960, calcium disodium ethylenediaminetetraacetate (CaNa2EDTA) com-
pletely replaced BAL. Beginning in 1960, combined therapy with BAL and CaNa2EDTA (Chisolm
1968) resulted in a very substantial reduction in mortality.
12-68
-------
Determining precise values for lead exposures necessary to produce acute symptoms, such
as lethargy, vomiting, irritability, loss of appetite, dizziness, etc., or later neurotoxic
sequelae in humans is difficult in view of the usual sparsity of data on environmental lead
exposure levels, period(s) of exposure, or body burdens of lead existing prior to manifesta-
tion of symptoms. Nevertheless, enough information is available to permit reasonable esti-
mates to be made regarding the range of blood lead levels associated with acute encephalo-
pathic symptoms or death. Available data indicate that lower blood lead levels among children
than among adults are associated with acute encephalopathy symptoms. The most extensive com-
pilation of information on a pediatric population is a summarization (National Academy of
Sciences, 1972) of data from Chisolm (1962, 1965) and Chisolm and Harrison (1956). This data
compilation relates occurrence of acute encephalopathy and death in children in Baltimore to
blood lead levels determined by the Baltimore City Health Department (using the dithizone
method) between 1930 and 1970. Blood lead levels formerly regarded as "asymptomatic" and
other signs of acute lead poisoning were also tabulated. Increased lead absorption in the
absence of detected symptoms was observed at blood lead levels ranging from 60 to 300 M9/dl
(mean = 105 ug/dl). Acute lead poisoning symptoms other than signs of encephalopathy were
observed from approximately 60 to 450 ug/dl (mean = 178 ug/dl). Signs of encephalopathy
(hyperirritability, ataxia, convulsions, stupor, and coma) were associated with blood lead
levels of approximately 90 to 700 or 800 M9/dl (mean = 330 ug/dl). The distribution of blood
lead levels associated with death (mean = 327 ug/dl) was essentially the same as for levels
yielding encephalopathy. These data suggest that blood lead levels capable of producing death
in children are essentially identical to those associated with acute encephalopathy and that
such effects are usually manifested in children starting at blood lead levels of approximately
100 ug/dl. Certain other evidence from scattered medical reports (Gant, 1938; Smith et al.,
1938; Bradley et al., 1956; Bradley and Baumgartner, 1958; Cumings, 1959; Rummo et al., 1979),
however, suggests that acute encephalopathy in the most highly susceptible children may be
associated with blood lead levels in the range of 80-100 ug/dl. These latter reports are
evaluated in detail in the 1977 EPA document Air Quality Criteria for Lead (U.S. EPA, 1977).
From the preceding discussion, it can be seen that severity of symptoms varies widely for
different adults or children at increasing blood lead levels. Some show irreversible CNS
damage or death at blood lead levels around 100 ug/dl, whereas others may not show any of the
usual clinical signs of lead intoxication even at blood lead levels in the 100-200 ug/dl or
higher range. This diversity of response may be due to the following: (1) individual bio-
logical variation in lead uptake or susceptibility to lead effects; (2) changes in blood lead
values from the time of initial damaging intoxication; (3) greater tolerance for a gradually
accumulating lead burden; (4) other interacting or confounding factors, such as nutritional
12-69
-------
state or inaccurate determinations of blood lead; or (5) lack of use of blind evaluation pro-
cedures on the part of the evaluators. It should also be noted that a continuous gradation of
frequency and severity of neurotoxic symptoms extends into the lower ranges of lead exposure.
Morphologica-1 findings vary in cases of fatal lead encephalopathy among children
(Blackman, 1937; Pentschew, 1965; Popoff et al., 1963). Reported neuropathologic findings are
essentially the same for adults and children. On macroscopic examination the brains are often
edematous and congested. Microscopically, cerebral edema, altered capillaries (endothelial
hypertrophy and hyperplasia), and peri vascular glial proliferation often occur. Neuronal
damage is variable and may be caused by anoxia. However, in some cases gross and microscopic
changes are minimal (Pentschew, 1965). Pentschew (1965) described neuropathology findings for
20 cases of acute lead encephalopathy in infants and young children. The most common finding
was activation of intracerebral capillaries characterized by dilation of the capillaries, with
swelling of endothelial cells. Diffuse astrocytic proliferation, an early morphological re-
sponse to increased permeability of the blood-brain barrier, was often present. Concurrent
with such alterations, especially evident in the cerebellum, were changes that Pentschew
(1965) attributed to hemodynamic disorders, i.e., ischemic changes manifested as cell
necrosis, perineuronal incrustations, and loss of neurons, especially in isocortex and basal
ganglia.
Attempts have been made to better understand brain changes associated with encephalopathy
by studying animal models. Studies of lead intoxication in the CNS of developing rats have
shown vasculopathic changes (Pentschew and Garro, 1966), reduced cerebral cortical thickness
and reduced number of synapses per neuron (Krigman et al., 1974a), and reduced cerebral axonal
size (Krigman et al., 1974b). Biochemical changes in the CNS of lead-treated neonatal rats
have also demonstrated reduced lipid brain content but no alterations of neural lipid composi-
tion (Krigman et al., 1974a) and a reduced cerebellar DNA content (Michaelson, 1973). in
cases of lower level lead exposure, subjectively recognizable neuropathologic features may not
occur (Krigman, 1978). Instead there may be subtle changes at the level of the synapse
(Silbergeld etal., 1980a) or dendritic field, myelin-axon relations, and organization of
synaptic patterns (Krigman, 1978). Since the nervous system is a dynamic structure rather
than a static one, it undergoes compensatory changes (Norton and Culver, 1977), maturation and
aging (Sotelo and Palay, 1971), and structural changes in response to environmental stimuli
(Coss and Glohus, 1978). Thus, whereas massive structural damage in many cases of acute en-
cephalopathy would be expected to cause lasting neurotoxic sequelae, some other CNS effects
due to severe early lead insult might be reversible or compensated for, depending upon age and
duration of toxic exposure. This raises the question of whether effects of early overt lead
intoxication are reversible beyond the initial intoxication or continue to persist.
12-70
-------
In cases of severe or prolonged nonfatal episodes of lead encephalopathy, there occur
neurological sequelae qualitatively similar to those often seen following traumatic or infec-
tious cerebral injury, with permanent sequelae being more common in children than in adults
(Mel 1 ins and Jenkins, 1955; Chi solm, 1962, 1968). The most severe sequelae in children are
cortical atrophy, hydrocephalus, convulsive seizures, and severe mental retardation (Mellins
and Jenkins, 1955; Perl stein and Attala, 1966; Chi solm, 1968). Children who recover from
acute lead encephalopathy but are re-exposed to lead almost invariably show evidence of per-
manent central nervous system damage (Chisolm and Harrison, 1956). Even if further lead expo-
sure is minimized, 25-50 percent show severe permanent sequelae, such as seizure disorders,
blindness, and hemiparesis (Chisolm and Barltrop, 1979).
Lasting neurotoxic sequelae of overt lead intoxication in children in the absence of
acute encephalopathy have also been reported. Byers and Lord (1943), for example, reported
that 19 out of 20 children with previous lead poisoning later made unsatisfactory progress in
school, presumably due to sensorimotor deficits, short attention span, and behavioral dis-
orders. These latter types of effects have since been confirmed in children with known high
exposures to lead, but without a history of life-threatening forms of acute encephalopathy
(Chisolm and Harrison, 1956; Cohen and Ahrens, 1959; Kline, 1960). Perlstein and Attala
(1966) also reported neurological sequelae in 140 of 386 children (37 percent) following lead
poisoning without encephalopathy. Such sequelae included mental retardation, seizures, cere-
bral palsy, optic atrophy, and visual-perceptual problems in some children with minimal intel-
lectual impairment. The severity of sequelae was related to severity of earlier observed
symptoms. For 9 percent of those children who appeared to be without severe symptoms at the
time of diagnosis of overt lead poisoning, mental retardation was observed upon later follow-
up. Since no control group was included in their study, one may question whether the neuro-
logical effects observed by Perlstein and Attala (1966) were persisting effects of earlier
overt lead intoxication without encephalopathy; however, it is extremely unlikely that 37 per-
cent of any randomly selected control group from the general pediatric population would exhi-
bit the types of neurological problems observed by Perlstein and Attala (1966).
Numerous studies (Cohen et al., 1976; Fejerman et al., 1973; Pueschel et al., 1972; Sachs
et al., 1978, 1979, 1982) suggest that, in the absence of encephalopathy, chelation therapy
may ameliorate the persistence of neurotoxic effects of overt lead poisoning (especially cog-
nitive, perceptual, and behavioral deficits). On the other hand, one recent study found a
residual effect on fine motor performance even after chelation (Kirkconnell and Hicks, 1980).
In summary, pertinent literature definitively demonstrates that lead poisoning with
encephalopathy results in a greatly increased incidence of permanent neurological and cogni-
tive impairments. Also, several studies further indicate that children with symptomatic lead
12-71
-------
poisoning in the absence of encephalopathy also show a later increased incidence of neurologi-
cal and behavioral impairments.
12.4.2.2.2 Non-overt lead intoxication in children. In addition to neurotoxic effects asso-
ciated with overt lead intoxication in children, substantial evidence indicates that lead ex-
posures not leading to overt lead intoxication in children can induce neurological dysfunc-
tions. This issue has attracted much attention and generated considerable controversy during
the past 10-15 years. However, the evidence for and against the occurrence of significant
neurotoxic deficits at relatively low levels of lead exposure has been quite mixed and largely
interpretable only after a thorough critical evaluation of methods employed in the various
important studies on the subject. Based on five of the criteria listed earlier (i.e., ade-
quate markers of exposure to lead, sensitive measures, appropriate subject selection, control
of confounding covariates, and appropriate statistical analysis), the population studies sum-
marized in Table 12-2 were conducted rigorously enough to warrant at least some consideration
here. Even so, no epidemiologies! study is completely flawless and, therefore, overall inter-
pretation of such findings must be based on evaluation of the following: (1) the internal
consistency and quality of each study; (2) the consistency of results obtained across inde-
pendently conducted studies; and (3) the plausibility of results in view of other available
information.
Rutter (1980) has classified studies evaluating neurobehavioral effects of lead exposure
in non-overtly lead intoxicated children according to several types, including four categories
reviewed below: (1) clinic-type studies of children thought to be at risk because of high
lead levels; (2) other studies of children drawn from general (typically urban or suburban)
pediatric populations; (3) samples of children living more specifically in close proximity to
lead emitting smelters; and (4) studies of mentally retarded or behaviorally deviant children
Major attention is accorded here to studies falling under the first three categories. A
final section discusses some initial results beginning to emerge from long-term prospective
studies, which attempt to relate effects on early neuropsychological development and later
neuropsychologic functioning to lead exposure histories for children documented back to birth
or even prenatally.
12.4.2.2.2.1 Clinic-type studies of children with high lead levels. The clinic-type
studies are generally typified by evaluation of children with relatively high lead body bur-
dens as identified through lead screening programs or other large-scale programs focusing on
mother-infant health relationships and early childhood development.
De la Burde and Choate (1972) observed neurological dysfunctions, fine motor dysfunction
impaired concept formation, and altered behavioral profiles in 70 preschool children exhibit-
ing pica and elevated blood lead levels (in all cases above 30 ug/dl; mean = 59 ug/dl) in com-
parison with matched control subjects not engaging in pica. Subjects were drawn from the
12-72
-------
TABLE 12-2. SUMMARY OF STUDIES ON NEUROBEHAVIORAL FUNCTIONS OF LEAD-EXPOSED CHILDREN9
tvj
I
Population
Reference studied N/group
Age at Blood lead,
testing, yr ug/dl
(range) (range or tS.D.)
Psychometri c
tests employed
Summa
ry of results
Levels of .
significance
Clinic-type studies of children with high lead levels
de la Burde and Inner city C = 72
Choate (1972) (Richmond, VA) Pb = 70
de la Burde and Follow-up C = 67
Choate (1975) same subjects Pb = 70
4.0 ?C d
4.0 58 (30-100)°
7-8 PbT: 112 ug/g
7-8 202 M9/a
IQ (Stanford-
Binet) %
Fine motor
Gross Motor
Concept formation
Behavior profile
WISC Full Scale
IQ %
Verbal IQ
Performance IQ
Bender-Gestalt
Reading
Spelling
Arithmetic
Score:
subnormal
Score:
subnormal
Goodenough-Harris draw test
Rummo (1974); Inner city C = 45
RUMO et al. (Providence, RI) Pbj = 15
(1979) Pb2 = 20
Pbs = 10
Kotok (1972) Inner city C = 25
(New Haven, CT) Pb = 24
Kotok et al. Inner city C = 36
(1977) (Rochester, NY) Pb = 31
5.8 23 (±8)
5.6 ,, flx 61 (±7)
5.6 *•* *; 68 (±13)
5.3 88 (±41)
2.7 (1.1-5.5) 38 (20-55)
2.8 (1.0-5.8) 81 (58-137)
3.6 (1.9-5.6) 28 (11-40)
3.6 (1.7-5.4) 80 (61-200)
Auditory vocal assoc
Tactile recognition
Behavior profile
McCarthy Scales:
Gen. cognitive
Verbal
Perceptual
Quantitative
Memory
Motor
Parent ratings
Neurologic exam
Denver Develop-
mental :
Gross Motor
Fine motor
Language
IQ Equivalent:
Social
Spatial
Spoken vocal.
Info-conprehersion
Visual attention
Auditory memory
.
C
93
46
48
45
47
52
8
7/12 i
Norm
1.00-
i.oo!
1.00
C
:e 10
26
7
10
10
e 90
:e 6
9
13
27
7
11
7
1
13
3
3
Pbj
94
46
49
44
46
52
10
teasures
C
1.02
0.82
0.82
C
126
101
93
96
93
100
Pb
89
25
45
16
15
30
87
24
18
24
49
12
16
12
13
31
15
25
Pb2
88
44
46
41
43
50
10
sig.
Pb
1.06
0.81
0.73
Pb
124
92
92
94
90
93
Pb3
77
37
38
35
36
40
18
<0.05
<0.05
N.S.
N.S.
<0.01
0.01
N.S.
N.S.
0.01
N.S.
N.S.
N.S.
0.02
0.01
0.05
0.001
<0.01
<0.05
<0.05
<0.01
<0.01
<0.01
<0.01
different
T
f
N.S.f
0.10
<0.10
>0.10
>0.10
>0.10
>0.10
-------
TABLE 12-2. (continued)
Age at
Population testing, yr
Reference studied N/group (range)
Perino and f Inner city Low Pb = 50 3-6
Ernhart (1974)r (New York, NY) Hod. Pb = 30 3-6
Ernhart at al. Follow-up same Low Pb = 31 8-13
(1981V subjects Nod. Pb = 32
1— »
r\3
i
•-j
.pi
General Population Studies
Needlewan et al. Urban C = 100 7
(1979)" (Boston, MA) Pb = 58 7
NcBride et al. Urban/suburban Low Pb = MOO 4/5
(1982) (Sydney, Australia) Hod. Pb = >100 4/5
Blood lead,
ug/dl Psychometric Levels of .
(range or ±S.O.) tests enployed Sunwary of results significance
10-30 McCarthy Scales:
40-70 Gen. cognitive
Verbal
Perceptual
Quantitative
Memory
Motor
21 (±4)9 McCarthy Scales:
32 (±5) Gen. cognitive
Verbal
Perceptual
Quantitative
Memory
Motor
Reading tests
Conners teacher ratings
Various experimental tests
£
PbT: <10 pp» W1SC Full Scale IQ
>20 pp* Verbal IQ
Performance IQ
Seashore Rhythm Test
Token Test
Sentence Repetition Test
Delayed Reaction Time C
Teacher Ratings
2-9 ug/dl Peabody Picture
19-29 g/dl Vocab. Test
Fine Motor Tracking C >
Pegboard
Tapping Test
Bean Walk
Standing Balance C >
Rutter Activity
Scale
Low Mod.
90 80
44 39
44 37
48 44
45 42
46 42
Low Hod.
94 82
48 41
43 40
43 38
44 39
49 46
Mot Reported
Not Reported
Not Reported
C Pb
106. 6 102. 1
103.9 99.3
108.7 104.9
21.6 19.4
24.8 23.6
12.6 11.3
> Pb on 3/4 blocks
9.5 8.2
Low Mod.
-105 -104
Pb 1/4 comparisons
-20 -20
-30 -31
-5 -4
Pb 1/4 comparisons
-1.9 -2.1
<0.01
<0.05
<0.05
N.S.
N.S.
N.S.
<0.05
<0.05
N.S.
N.S.
N.S.
<0.05
N.S.
N.S.
N.S.
0.03
0.06
0.12
0.002
0.09
0.04
<0.01
0.02
N.S.
<0.05
N.S.
N.S.
N.S.
<0.05
N.S.
-------
TABLE 12-2. (continued)
Reference
Yule et al.
(1981)
Yule et al.
(1984)
Population
studied
Urban
(London, England)
Same subjects
in Yule et al.
(1981)
Age at
testing, yr
N/group (range)
Group
Group
Group
Group
1 = 34 9
2 — 4o 9 /c-.i9\
3 = 49 8 l6 U)
4 = 35 8
Same Same
Blood lead,
tig/dl
(range or ±S.O.)
8.8J (7-10)
11.6
14.5
19.6
(11-12)
(13-16)
(17-32)
Same
Psychometric
tests employed
Summary
Group: 1
WISC-R Full Scale IQ
Verbal IQ
Performance IQ
Vernon Spelling Test
Vernon Hath Test
Neale Reading Accuracy
Neale Reading Compre.
Needleman Teacher
Ratings
Conners Teacher
t*_i. • f
103
101
106
104
t 97
* 121
117
1.53
(4/11
0.
^ tm .*_ _
2
103
101
103
98
97
110
110
1.54
items
26
Levels of b
of results significance
3
96
95
98
92
95
96
95
2.45
4
96
94
99
89
95
89
88
2.63
0.027
0.043
0.102
0.001
N.S.
0.001
0.001
0.096
sig. different)
0.
37
jf\ /It \
0.04
ro
i
Lansdown et al. Urban
(1986) (London, England)
Low = 80
High = 82
7-12
13-24
Smith et al. Urban High = 155
(1983) (London, England) Med = 103
Low = 145
6,7
6,7
6,7
PbT £ 8.0
PbT = 5-5.5
PbT < 2.5
£A11 in M9/8)
X PbB = 13.1
Ratings (3/4 factors sig. at p <0.05)
Rutter Teacher (2/26 items sig. at p SO.05)
Ratings, including (5/26 items differ at 0.05
-------
TABLE 12-2. (continued)
l\3
I
CTi
Reference
Harvey et al.
Age at Blood lead,
Population testing, yr ug/dl
studied N/group (range) (range or ±S.D. )
Urban 189 2.5 15.5 (6-30)
(1983, 1984) (Birmingham, England)
Silva et al. (1986b)
Schroeder et al.
(1985)
Schroeder and
Hawk (1986)
Smelter Area Studies
Landrigan et al.
(1975)
Urban 579 11 11.1 (4-50)
(Dunedin, New Zealand)
Rural/urban 104 <2.5 or >2.5 6-59
(Wake County, (0.8-6.5)
North Carolina)
Follow-up same 50 6-12 S30
subjects
Rural /urban 75 2 21 (6-47)
(Lenoir and
Hanover Counties,
North Carolina
Smelter area C = 78 9.3 ,, ft,, „, <40
(El Paso, TX) Pb=46 8.3 lJ'e "•*' 40-68
Psychometric
tests employed
British Ability Scales
Naming
Recall
Comprehension
Recognition
IQ
Stanford-Binet Items
Shapes
Blocks
Beads
Playroom Activity
WISC-R Full Scale IQ
Performance IQ
Verbal
Rutter Behavior Rating
Parent
Teacher
Inattention Rating
Parent
Teacher
Hyperactivity Rating
Parent
Teacher
Burt Reading Test
Levels of t
S unwary of results significance
Regression F Ratio
<1
1.26
<1
<1
<1
<1
2.34
2.46
?
r = -0.06
= -0.03
= -0.05
R2 Incrs. by Pb
D~ZI 1.38
0.10 1.23
0.24 0.26
0.25 1.51
0.12 1.01
0.12 0.82
0.43 0.28
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
0.003
0.008
N.S.
0.001
0.015
0.028
N.S.
Bayley HOI or Stanford- Regression analysis
Binet
Stanf ord-Bi net
Stanford-Binet
WISC Full Scale IQ"
WPPSI Full Scale IQ
WISC + WPPSI Combined
WISC + WPPSI Subscales
Neurologic testing
of sources for IQ
effect:
Lead: F = 7.689
SES: F = 20.159
Lead: F <1
Regression analysis of
IQ against:
Current PbB: F = 12.31
Max. Pb6: F = 10.55
Mean PbB: F = 10.08
C Pb
93 89
91 86
93 88
C > Pb on 13/14 scales
7/14 scales sig. different
C > Pb on 7/8 tests
1/8 tests sig. different
<0.01
<0.001
N.S.
<0. 0008
<0.0018
<0.002
N.S.
N.S.
N.S.
<0.05
<0.01
-------
TABLE 12-2. (continued)
Age at
Population testing, yr
Reference studied N/group (range)
McNeil and Ptasnik Smelter area C = 37 9 ,, a ,„.,
(1975) (El Paso, TX) Pb = 101 9 1I-S'-U}J
Ratcliffe (1977) Smelter area Mod. Pb =23 4.7 (4.1-5.6)
(Manchester, Hi Pb = 24 4.8 (4.2-5.4)
England)
Winneke et al. Smelter area C = 26 8
(1982a) (Duisburg, FRG) Pb = 26 8
Winneke et al. Smelter area 89 9.4
(1983) (Stolburg, FRG)
Blood lead,
ug/dl Psychometric Levels of b
(range or iS.D. ) tests employed Summary of results significance
29 (14-39) McCarthy General
58 (40-93) Cognitive
WISC-WAIS Full Scale
IQ
Oseretsky Motor Level
California Person-
ality C >
Frostig Perceptual
Quotient
Finger-Thumb
Apposition
28 (18-35) Griffiths Mental Dev.
44 (36-64) Frostig Visual
Perception
Pegboard Test
Dominant hand
Nondominant hand
L
PbT = 2.4 ppm German WISC Full Scale
PbT =9.2 ppm Verbal IQ
No PbB Performance IQ
Bender Gestalt Test
Standard Neurological Tests
Conners Teacher Ratings
C Pb
82 81
89 87
101 97
Pb, 6/10 items
100 103
27 29
Mod. High
108 102
14.3 11.8
17.5 17.3
19.5 19.8
C Pb
122 117
130 124
130 123
17.2 19.6
2.7 7.2
? ?
N.S.
N.S.
N.S.
<0.05
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
<0.05
N.S.
N.S.
PbT: 6.16 ppmh German WISC % Variance Due to PbT
PbB: 14.3 M9/dl Full Scale IQ
Verbal IQ
Performance IQ
Bender Gestalt Test
Standard Neurological Tests
Conners Teacher Ratings
Wiener Reaction Performance
-0.0
-0.5
+0.6
+2.1
+1.2
0.4-1.3
+2.0
N.S.
N.S.
N.S.
<0.05
N.S.
N.S.
N.S.
-------
TABLE 12-2. (continued)
Reference
Winneke et al.
(1984)
Age at Blood lead,
Population testing, yr M9/dl Psychometric
studied N/group (range) (range or ±S.D. ) tests employed
Summary of results
Levels of b
significance
Smelter area 122 6.5 8.2(4.4-22.8) German WISC % Variance Due to Pb
,(Nordenham, FRG) Short form
Verbal IQ
Performance
Bender Gestalt Test
Signalled Reaction Time
Short
Long
Wiener Reaction Tine
Easy
Difficult
-0.3
+0.3
-2.4
+0.5
+0.1
-0.2
+4.3
+11.0
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
<0.05
<0.01
Abbreviations: C = control subjects; Pb = lead-exposed subjects; MDI = mental development index; N.S. = nonsignificant (p >0.05); PbT = tooth lead;
,_. WISC = Wechsler Intelligence Scale for Children; WPPSI = Wechsler Preschool and Primary Scale of Intelligence; RT = Reaction Time.
i Significance levels are those found after partial ing out confounding covariates.
oocUrinary coproporphyrin levels were not elevated.
Some with positive radiologic findings, suggesting earlier exposure in excess of 40-60 ug/dl.
ePercent of each group scoring "borderline," "suspect," "defective," or "abnormal."
Reanalysis of data by Ernhart correcting for methodological problems in earlier published analyses described here mainly did not substantiate significant
differences between control and Pb-exposed children indicated in last two columns to the right (see chapter text).
9Dentine levels not reported for statistical reasons.
Reanalyses of Needleman data correcting for methodological problems in earlier published analyses confirmed significant differences between study groups
indicated in last two righthand columns for WISC IQ test results (see chapter text).
^Hain measure was dentine lead (PbT).
•*Blood lead levels taken 9-12 months prior to testing; none above 33 ug/dl.
TJata not corrected for age.
This F ratio is result of testing the difference in sums of squares for two regression equations (one including and one excluding blood lead level as an
independent variable) against the residual nean square of the equation including blood lead.
TJsed for children over 5 years of age.
"Used for children under 5 years of age.
-------
Collaborative Study of Cerebral Palsy, Mental Retardation, and Other Neurologic Disorders of
Infancy and Childhood (Broman et al., 1975), which was conducted in Richmond, Virginia, and
had a total population of 3400 mothers. All mothers in this group were followed throughout
pregnancy and all children were postnatally evaluated by regular pediatric neurologic examina-
tions, psychological testing, and medical interviews. All children subject to prenatal, peri-
natal, and early postnatal insults were excluded from the study, and all had to have normal
neurologic examinations and Bayley tests at eight to nine months of age. These are important
points which add value to the study. It is unfortunate that blood lead data were not regu-
larly obtained; however, at the time of the study in the late 1960s, 10-20 ml of venous blood
was required for a blood lead determination and such samples usually had to be obtained by
either jugular or femoral puncture. The other control features (housing location and repeated
urinary coproporphyrin tests) would be considered the state of the art for such a study at the
time that it was carried out.
In a follow-up study on the same children (at 7-8 years old), de la Burde and Choate
(1975) reported continuing CMS impairment in the lead-exposed group as assessed by a variety
of psychological and neurological tests. In addition, seven times as many lead-exposed
children were repeating grades in school or being referred to the school psychologist, despite
many of their blood lead levels having by then dropped significantly from the initial study.
In general, the de la Burde and Choate (1972, 1975) studies appear to be methodologically
sound, having many features that strengthen the case for the validity of their findings. For
example, there were appreciable numbers of children (67 lead-exposed and 70 controls) whose
blood lead values were obtained in preschool years and who were old enough (7 years) during
the follow-up study to cooperate adequately for reliable psychological testing. The psycho-
metric tests employed were well standardized and acceptable as sensitive indicators of neuro-
behavioral dysfunction, and the testing was carried out in a blind fashion (i.e., without the
evaluators knowing which were control or lead-exposed subjects).
The de la Burde and Choate (1972, 1975) studies might be criticized on several points,
but none provide sufficient grounds for rejecting their results. One difficulty is that blood
lead values were not determined for control subjects in the initial study; but the lack of
history of pica for paint and plaster, as well as tooth lead analyses done later for the
follow-up study, render it improbable that appreciable numbers of lead-exposed subjects might
have been wrongly assigned to the control group. Subjects in the control group did have a
history of pica, but not for paint. Also, results indicating no measurable coproporphyrins in
the urine of control subjects at the time of initial testing further confirm proper assignment
of those children to the nonexposed control group. A second point of criticism is the use of
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multiple chi-square statistical analyses, but the fact that the control subjects did signifi-
cantly better on virtually every measure makes it unlikely that all of the observed effects
were due to chance alone. One last problem concerns ambiguities in subject selection which
complicate interpretation of the results obtained. Because the lead-exposed group included
children with blood lead levels of 40-100 ug/dl, or of at least 30 ug/dl with "positive radio-
graphic findings of lead lines in the long bones, metallic deposits in the intestines, or
both," observed deficits might be attributed to blood lead levels as low as 30 ug/dl. Other
evidence (Betts et al., 1973), however, suggests that such a simple interpretation is probably
not accurate. That is, the Betts et al. (1973) study indicates that lead lines are usually
seen only if blood levels exceed 60 ug/dl for most children at some time during exposure
although some (about 25 percent) may show lead lines at blood lead levels of 40-60 ug/dl. in
view of this, the de la Burde and Choate results can probably be most reasonably interpreted
as showing persisting neurobehavioral deficits at blood lead levels of 40-60 ug/dl or higher.
In another clinic-type child study, Rummo (1974) and Rummo et al. (1979) found signifi-
cant neurobehavioral deficits (hyperactivity, lower scores on McCarthy scales of cognitive
function, etc.) among Providence, Rhode Island, inner-city children who had previously exper-
ienced high levels of lead exposure that had produced acute lead encephalopathy. Mean maximum
blood lead levels recorded for those children at the time of encephalopathy were 88 ± 40
ug/dl. However, children with moderate blood lead elevation but not manifesting symptoms of
encephalopathy were not significantly different (at p <0.05) from controls on any measure of
cognitive functioning, psychomotor performance, or hyperactivity. Still, when the data from
the Rummo et al. (1979) study for performance on the McCarthy General Cognitive Index or
several McCarthy Subscales are compared (see Table 12-2), the scores for long-term moderate-
exposure subjects consistently fall below those for control subjects and lie between the
latter and the encephalopathy group scores. Thus, it appears that long-term moderate lead
exposure, in fact, likely exerted dose-related neurobehavioral effects. The overall dose-
response trend might have been shown to be statistically significant if other types of
analyses were used, if larger samples were assessed, or if control subjects were restricted to
blood lead values below 10 ug/dl. However, control for confounding variables in the different
exposure groups would also have to be considered. Note that (1) the maximum blood lead levels
for the short-term and long-term exposure subjects were all greater than 40 ug/dl (means = 61
± 7 and 68 ± 13 (jg/dl, respectively), whereas control subjects all had blood lead levels below
40 ug/dl (mean = 23 ± 8 ug/dl), and (2) the control and lead-exposed subjects were inner-city
children well matched for socioeconomic background, parental education levels, incidence of
pica, and other pertinent factors, but parental IQ was not ascertained and controlled for as a
potentially confounding variable.
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A somewhat similar pattern of results emerged from a study by Kotok et al. (1977) in
which 36 Rochester, New York, control-group children with blood lead levels less than 40 ug/dl
were compared with 31 children having distinctly elevated blood lead levels (61-200 pg/dl) but
no classical lead intoxication symptoms. Both groups were well matched on important back-
ground factors, notably including their propensity to exhibit pica. Again, no clearly statis-
tically significant differences between the two groups were found on numerous tests of cogni-
tive and sensory functions. However, mean scores of control-group children were consistently
higher than those of the lead-exposed group for all six of the ability classes listed, even
though the control group included children that had notably elevated blood lead values by
current medical standards. Kotok (1972) had reported earlier that developmental deficiencies
(using the comparatively insensitive Denver Development Screening test) in a group of children
having elevated lead levels (58-137 ug/dl) were identical to those in a control group similar
in age, sex, race, environment, neonatal condition, and presence of pica, but whose blood lead
levels were lower (20-55 ug/dl). Children in the lead-exposed group, however, had blood lead
levels as high as 137 ug/dl, whereas some control children had blood lead levels as high as 55
ug/dl. Thus, the study essentially compared two groups with different degrees of markedly
elevated lead exposure rather than one of lead-exposed versus nonexposed control children.
Perino and Ernhart (1974) reported a relationship between neurobehavioral deficits and
blood lead levels ranging from 40 to 70 ug/dl in a group of 80 inner-city preschool black
children, based on the results of a cross-sectional study including children detected as
having elevated lead levels via the New York City lead screening program. One key result
reported was that the high-lead children had McCarthy Scale IQ scores markedly lower than
those of the low-lead group (mean IQ = 80 versus 90, respectively). Also, the normal correla-
tion of 0.52 between parents' intelligence and that of their offspring was found to be reduced
to only 0.10 in the lead-exposed group, presumably because of the influence of another factor
(lead) that interfered with the normal intellectual development of the lead-exposed children.
Another possible explanation for the reported results, however, might be differences in the
educational backgrounds of parents of the control subjects when compared with lead-exposed
subjects, because parental education level was found to be significantly negatively related to
blood lead levels of the children participating in the Perino and Ernhart (1974) study. The
importance of this point lies in the fact that several other studies (McCall et al., 1972;
Elardo et al., 1975; Ivanans, 1975) have demonstrated that higher parental education levels
are associated with more rapid development and higher intelligence quotients (IQs) for their
children.
Ernhart et al. (1981) were able to trace and carry out follow-up evaluations on 63 of the
80 preschool children of the Perino and Ernhart (1974) study once they reached school age,
using the McCarthy IQ scales, various reading achievement tests, the Bender-Gestalt test, the
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Draw-A-Child test, and the Conners Teacher's Questionnaire for hyperactivity. The children's
blood lead levels were reported to be significantly correlated with FEP (r = 0.51) and dentine
lead levels (r = 0.43), but mean blood lead levels of the moderately elevated group had de-
creased after five years. When control variables of sex and parental IQ were extracted by
multivariate analyses, the observed differences were reported to be greatly reduced but
remained statistically significant for three of seven tests on the McCarthy scales in relation
to concurrently measured blood lead levels but not in relation to the earlier blood lead
levels or dentine lead levels for the same children. This led Ernhart et al. (1981) to re-
interpret their 1974 (Perino and Ernhart, 1974) IQ results (in which they had not controlled
for parental education) as either not likely being due to lead or, if due to lead, then repre-
senting only minimal effects on intelligence.
The Perino and Ernhart (1974) and Ernhart et al. (1981) studies were evaluated by an
expert committee convened by EPA in March, 1983. The committee reported (Expert Committee on
Pediatric Neurobehavioral Evaluations, 1983) certain methodological problems associated with
the analyses published by Perino and Ernhart (1974) and Ernhart et al. (1981). The committee
further recommended that the Ernhart data set be reanalyzed to deal with the methodological
problems. Results of reanalyses of the data have been submitted by Ernhart (1983, 1984-
Ernhart etal., 1985). Reanalysis of relationships between preschool-age children's blood
lead levels and concurrently obtained McCarthy Scales scores (which included corrections of
errors made in the earlier, published analyses for certain data calculations and degrees of
freedom used to determine statistical significance) revealed no statistically significant dif-
ferences (at p <0.05) due to lead; however, lower scores for the higher lead exposure group on
the General Cognitive Index (GCI) did approach significance at p <0.09. Also, reanalysis of
relationships between preschool lead levels and 5-year later school-age outcome variables
yielded no indication of persisting lead effects in terms of reading test results or scores on
the McCarthy GCI or most of the McCarthy Subscales (except for a p-value of 0.10 obtained for
Verbal Index scores). The reanalysis of relationships between school-age blood lead levels
(newly corrected for hematocrit variation effects) and concurrent reading test and McCarthy
Scales scores only found significant differences attributable to lead for lower McCarthy
Verbal Index scores (p <0.036 with a "deviant case" included in the analysis and p <0.07 with
the case excluded). Similar results were obtained with a different analysis employing a "lead
construct index" as a measure of lead exposure which combined preschool and school-age blood
lead levels and free erythrocyte protoporphyrin levels. Based on these results, Ernhart
et al. (1985) concluded that "the reanalyses provide no reasonable support for an interpreta-
tion of lead effects in these data." However, she also noted that it is recognized that there
was a certain level of unreliability in the measures used and that the sample size limited the
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power of the statistical analyses. Given such limitations and extensive attention accorded to
statistical control of potentially confounding variables in the reanalyses, it is notable that
an association between lead and lower Verbal Index scores was nevertheless observed across
several of the analyses (at p values ranging from <0.04 to 0.10) and that an association
between preschool lead levels and General Cognitive Index scores approached significance at
p <0.09. These observations (possibly due to chance alone from among the large number of
statistical analyses conducted) do not provide much evidence for associations between neuro-
psychologic deficits and lead exposures at the levels experienced by children in the Ernhart
study population; conversely, however, results of the reanalyses do not allow for a definitive
conclusion of "no-effect," either (as noted by Ernhart, 1983).
Other investigators (Shapiro and Marecek, 1984; Marecek et al., 1983) studied relation-
ships between lead exposures and psychometric testing outcomes among black children who had
been members of the Philadelphia Collaborative Perinatal Project (CPP), which included mainly
families of low socioeconomic status. From among a large target sample of eligible children
(those young enough to have deciduous teeth and no past history of head trauma, mental retar-
dation, or lead poisoning) invited to participate in the study (2,568 letters of invitation
were mailed), 199 families enrolled their children. Each child was scheduled for neuropsycho-
logic testing immediately following the loss of a tooth; primary and/or circumpulpal dentine
lead levels from shed deciduous teeth (mainly molars) were employed to provide an index of
lead exposure for the 188 children (aged 10.6 to 14.7 yr; X = 11.8 yr) who underwent neuropsy-
chologic testing. Data on socioeconomic status and several other potentially confounding
variables were obtained from CPP records, and IQ scores were obtained for the parents of a
subset of the children studied. Data analyses (hierarchical multiple regression analyses)
first evaluated relationships between dentine lead exposure indices and test scores obtained
several years earlier (at age 7 yr) on the Bender-Gestalt, Wechsler Intelligence Scale for
Children (WISC) subtests, and certain other neuropsychologic tests; analyses were also per-
formed using dentine lead data and results from concurrently administered psychometric tests.
For the age-seven tests, significant associations were reported between dentine lead and per-
formance IQ scores, but not for WISC verbal IQ scores. Similarly, significant relationships
(at p <0.05) were reported between dentine lead values and concurrently obtained test results
for performance abilities on the Bender-Gestalt, WISC, and other tests but not for verbal
abilities. This study, while qualitatively suggesting lead may affect performance abilities,
suffers from several methodological problems, including inadequate control for sampling bias,
retrospective estimation of age-seven lead exposure levels, poor control of covarying social
factors, and inadequate control for parental IQ influences for all children studied.
Odenbro et al. (1983) studied psychological development of children (aged 3-6 yr) seen in
Chicago Department of Health Clinics (August, 1976 - February, 1977), evaluating scores on the
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Denver Developmental Screening test and two subtests of the Wechsler Preschool and Primary
Scale of Intelligence (WPPSI) in relation to blood lead levels obtained by repeated sampling
during the three previous years. A significant correlation (r = -0.435, p <0.001) was re-
ported between perceptual visual-motor ability and mean blood lead levels. Statistically sig-
nificant (p <0.005) deficits in verbal productivity and perceptual visual motor performance
(measured by the WPPSI) were found for groups of children with mean blood lead levels of
30-40 |jg/dl and 40-60 ug/dl versus control children with mean blood lead levels <25 ug/dl
using two-tailed Student's t-tests. On the other hand, significant associations (p <0.05)
between blood lead levels and developmental retardations in language and fine-motor functions
were found only for the 40-60 ug/dl group, using the Denver Development Screening test and
chi-square analyses. These results are most clearly suggestive of neuropsychologic deficits
being associated with blood lead levels of 40-60 ug/dl in preschool children. However, par-
ental IQs were not measured and questions can be raised regarding the adequacy of the statis-
tical analyses employed, especially in regard to lack of use of multivariate analyses that
sufficiently control for confounding covariates such as parental education and socioeconomic
status.
In another study (Molina etal., 1983), high-risk children from families making lead-
glazed pottery in a Mexican village were evaluated for lead-associated neuropsychologic defi-
cits, using an appropriately adapted Spanish language version of the revised WISC (WISC-R)
test and the Bender-Gestalt test. Test results for 33 high-lead children (X age: 10 yr, 7
mo ± 2 yr, 7 mo) randomly selected from 64 school children with blood lead levels above 4Q
ug/dl (X: 63.4 ± 15.8 ug/dl) were compared with those for 30. lower lead children (X age:
10 yr, 2 mo ± 2 yr, 6 mo) with blood lead levels below 40 ug/dl (X: 26.3 ±8.0 ug/dl), using
the two-tailed Student's t-test and the Mann-Whitney U test. The high-lead children were
found to have significantly lower WISC-R full-scale IQ (p <0.01), verbal IQ (p <0.01), and
performance IQ (p <0.025) than did the low-lead control children, who were drawn from among
the same low socioeconomic class families as the high-lead children. A significant negative
linear correlation was also observed for the same categories of test scores among the high-
lead children, but not for such scores among the low-lead children. These results, highly
suggestive of lead-related neuropsychologic deficits in children with blood lead values over
40 ug/dl, must be viewed with caution in light of the failure to include parental IQ levels
and the lack of multivariate statistical analyses that explicitly controlled for age, sex, or
other confounding factors.
In summary, the studies reviewed above generally found that high-risk lead-exposure
groups did more poorly on IQ or other types of psychometric tests than referent control groups
with distinctly lower lead exposures. It is true that many of the studies did not control for
important confounding variables or, when such were taken into account, differences between
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lead-exposed and control subjects were reduced and, at times, often no longer statistically
significant. Still, the consistency of finding lower IQ values and other types of neuropsy-
chologic deficits among at-risk higher lead exposure children across most of the studies re-
viewed lends credence to cognitive deficits occurring in apparently asymptomatic children with
markedly elevated blood lead levels (i.e., starting at 40-60 ug/dl and ranging upwards to
70-80 ug/dl and higher values).
The magnitude of lead's effects on IQ at the high exposure levels evaluated in these
studies is difficult to estimate precisely due to variations in measurement instruments used,
variations in the extent to which various confounding factors were controlled for in the sta-
tistical analyses, and the fact that many of the referent control groups tended to have what
are now recognized to be elevated blood lead levels (i.e., averaging in the 20-40 ug/dl
range). Focusing on estimates of full-scale IQ deficits, Rummo (1974; Rummo et al., 1979)
observed a decrement of approximately 16 IQ points on the McCarthy GCI for postencephalopathic
children with blood lead values exceeding 80 M9/dl. Asymptomatic children with long-term lead
exposures yielding mean blood lead values of 68 pg/dl experienced an average 5-point IQ (GCI)
decrement, whereas short-term lead-exposed subjects with blood lead levels around 60 |jg/dl
showed no decrement compared to controls. The de la Burde subjects, with blood lead levels
averaging 58 ug/dl, had a mean Stanford-Binet IQ decrement of 5 points upon first testing (de
la Burde and Choate, 1972) and 3 points upon follow-up testing several years later (de la
Burde and Choate, 1975). Ernhart originally reported an average 10 point IQ (GCI) decrement
for children with blood lead values in the 40-70 pg/dl range upon first testing (Perino and
Ernhart, 1974) and 12 points upon follow-up 5 years later (Ernhart et al., 1981). However,
these reported large decrements appear to be due in part to confounding by uncontrolled
covariates in the original published data calculations and, upon reanalysis of the data (with
better control for confounding variables and with errors corrected), are apparently notably
reduced, although the amount of the reduction was not clearly specified in the submitted
reanalyses. While it could be argued that the Rummo and de la Burde decrements would also be
reduced in size if better control for confounding variables were employed, use of control
subjects with lower lead exposures (e.g., <10 |jg/dl) could also logically be expected to re-
sult in offsetting influences on IQ. Thus, it seems warranted to conclude that the average
decrements of about 5 IQ points observed in the de la Burde and Rummo studies represent a
reasonable estimate of the magnitude of full-scale IQ decrements associated with notably
elevated blood lead levels (X = 50-70 ug/dl) in asymptomatic children.
12.4.2.2.2.2 General population studies. These studies evaluated samples of non-overtly
lead intoxicated children drawn from and thought to be representative of the general pediatric
population. They generally aimed to evaluate asymptomatic children with lower lead body bur-
dens than those of high-risk children evaluated in most of the above clinic-type studies.
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A pioneering general population study was reported by Needleman et al. (1979), who used
shed deciduous teeth to index lead exposure. Teeth were donated from 70 percent of a total
population of 3329 first and second grade children from two towns near Boston. Almost all
children who donated teeth (2146) were rated by their teachers on an 11-item classroom be-
havior scale devised by the authors to assess attention disorders. An apparent dose-response
function was reported for ratings on the behavior scale, not taking potentially confounding
variables into account. After excluding various subjects for control reasons, two groups
(<10th and >90th percentiles of non-circumpulpal dentine lead levels) were provisionally
selected for further in-depth neuropsychologic testing. Later, some provisionally eligible
children were also excluded for various reasons, leaving 100 low-lead (<10 ppm dentine lead)
children for comparison with 58 high-lead (>20 ppm dentine lead) children in statistical
analyses reported by Needleman et al. (1979). A preliminary analysis on 39 non-lead variables
showed significant differences between the low- and high-lead groups for age, maternal IQ and
education, maternal age at time of birth, paternal SES, and paternal education. Some of these
variables were entered as covariates into an analysis of covariance along with lead. Signifi-
cant effects (p <0.05) were reported for full-scale WISC-R IQ scores, WISC-R verbal IQ scores,
for 9 of 11 classroom behavior scale items, and several experimental measures of perceptual-
motor behavior.
Additional papers published by Needleman and coworkers have reported results of the same
or further analyses of the data discussed in the initial paper by Needleman et al. (1979).
For example, a paper by Needleman (1982) provided a summary overview of findings from the
Needleman et al. (1979) study and findings reported by Burchfiel et al. (1980) that are dis-
cussed later in Section 12.4.2.2.2.7 concerning EEG patterns for a subset of children included
in the 1979 study. Needleman (1982) summarized results of an additional analysis of the 1979
data set reported elsewhere by Needleman et al. (1982). More specifically, cumulative fre-
quency distributions of verbal IQ scores for low- and Mgh-lead subjects from the 1979 study
were reported by Needleman et al. (1982), and the key point made was that the average IQ
deficit of four points demonstrated by the 1979 study did not just reflect children with al-
ready low IQs having their cognitive abilities further impaired. Rather, the entire distribu-
tion of IQ scores across all IQ levels was reported to be shifted downward in the high-lead
group, with none of the children in that group having verbal IQs over 125. Another paper, by
Bellinger and Needleman (1983), provided still further follow-up analyses of the original
(Needleman et al., 1979) data set, focusing mainly on comparison of the low- and high-lead
children's observed versus expected IQs based on their mother's IQ. Bellinger and Needleman
reported that regression analyses showed that IQs of children with elevated levels of dentine
lead (>20 ppm) fell below those expected based on their mothers' IQs and that the amount by
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which a child's IQ fell below the expected value increased with increasing dentine lead levels
in a nonlinear fashion. Scatter plots of IQ residuals by dentine lead levels, as illustrated
and discussed by Bellinger and Needleman (1983), indicated that regressions for the control
children with dentine lead below 10 ppm and for high-lead children with 20-29.9 ppm dentine
lead did not reveal significant associations between increasing lead levels in that range and
IQ residuals. This is in contrast to statistically significant (p <0.05) correlations found
between IQ residuals and dentine lead for high-lead group children with 30-39.9 ppm dentine
lead levels.
The Needleman et al. (1979) study and spin-off analyses published later by Needleman and
coworkers were critically evaluated by the same Expert Committee on Neurobehavioral Evalua-
tions noted above that was convened by EPA in March, 1983, to evaluate the Peri no and Ernhart
(1974) and Ernhart et al. (1981) studies. The Committee's report (Expert Committee, 1983)
noted methodological problems with certain of the the published analyses and findings reported
by Needleman et al. (1979) or in subsequent papers by Needleman and coworkers concerning addi-
tional analyses of the same data set. The Committee also recommended that the Needleman data
set be reanalyzed. Reanalyses carried out in response to the Committee's recommendations have
been reported by Needleman (1984), Needleman et al. (1985), and U.S. EPA's Office of Policy
Analysis (1984) as confirming the published findings on significant associations between
elevated dentine lead levels and decrements in IQ, after correcting errors in data calcula-
tions detected in earlier published analyses and using alternative model specifications that
incorporated better control for potentially confounding factors.
The average magnitude of the full-scale IQ decrement attributable to lead was estimated
in the original published Needleman analyses to be about 4 points after control for confound-
ing factors. Based upon the reanalyses submitted, the size of the full-scale lead effect
appears to remain about the same (i.e., around 4 points) after controlling for confounding
variables. It is, however, extremely difficult to define with confidence quantitative dose-
response relationships based on the Needleman data, beyond the statement that average IQ de-
crements of about 4.0 points appear to be associated with lead exposure levels experienced by
the Needleman high-lead group. Among that group, statistically significant (p <0.05) IQ de-
crements appear to remain (after controlling for confounding variables) for children with
30-39.9 ppm dentine lead levels, but not for children with 20-29.9 ppm or lower dentine lead
levels, as reported by Bellinger and Needleman (1983). Only limited data exist by which one
might attempt to estimate blood lead values likely associated with the observed IQ effects;
and the available information points broadly toward an average blood lead concentration in the
30-50 ug/dl range. An average 4-point full-scale IQ decrement associated with average blood
lead values in that range would be consistent with the mean 5-point decrement estimated
earlier to occur at somewhat higher average blood lead levels of 50-70 pg/dl.
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Recently, Bellinger et al. (19845) followed up on the academic performance of a subset
of the children initially evaluated by Needleman et al. (1979). Of the 118 first and second
grade children who were classified into low (<10 ppm) and elevated (£20 ppm) dentine lead
groups by Needleman et al. (1979), 70 were available for study 4 years later. In addition,
71 children with midrange tooth lead levels (10.0-19.9 ppm) were included in the follow-up In-
vestigation. Contemporary blood lead levels could not be obtained. Four types of outcome
measures were assessed: (1) standardized IQ measures, viz., the most recently available
scores for the Otis-Lennon Mental Ability Test, as routinely administered by the school
system; (2) teacher ratings, comprising a 24-item pupil-rating scale and the same 11-1tea
scale used by Needleman et al. (1979); (3) indices of school failure, i. e., remedial In-
struction or grade retention; and (4) direct observation of classroom behavior patterns re-
flecting inattention, distractibility, etc. Various statistical analyses suggested that only
grade retention was clearly associated with past dentine lead levels; other outcomes tended to
be in the predicted direction of effect but generally at p values between 0.05 and 0.15. Of
some note is the fact that the teacher rating scale revealed no effect of lead, a finding that
contrasts with earlier results of Needleman et al. (1979) and a more recent replication
(albeit without control for social factors) by Yule et al. (1984).
A study of urban children in Sydney, Australia (McBride et al., 1982) involved 454 pre-
schoolers (aged 4-5 yr) with blood lead levels of 2-29 ug/dl. Children born at the Women's
Hospital in Sydney were recruited via personal letter. No blood lead measures were available
on non-participants. Blood levels were evaluated at the time of neurobehavioral testing, but
earlier exposure history was apparently not assessed. Using a multiple statistical comparison
procedure and Bonferroni correction to protect against study-wise error, no statistically sig-
nificant differences were found between two groups with blood lead levels more than one stand-
ard deviation above and below the mean (>19 ug/dl versus <9 ug/dl) on the Peabody Picture
Vocabulary IQ Test, on a parent rating scale of hyperactivity devised by Rutter, or on three
tests of motor ability (pegboard, standing balance, and finger tapping). In one test of fine
motor coordination (tracking), five-year old boys in the higher lead group performed worse
than boys in the lower lead group. In one test of gross motor skill (walking balance), results
for the two age groups were conflicting. This study suffers from many methodological weak-
nesses and cannot be regarded as providing evidence for or against an effect of low-level lead
exposures in non-overtly lead intoxicated children. For example, a comparison of socio-
economic status (father's occupation and mother's education) of the study sample with the
general population showed that it was higher than Bureau of Census statistics for the
Australian work force as a whole. Also, there was apparently some self-selection bias due to
a high, proportion of professionals living near the hospital, and certain other important demo-
graphic variables, such as mother's IQ, were not evaluated.
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Another recent large-scale study (Smith et al., 1983) of tooth lead, behavior, intelli-
gence, and a variety of other psychological skills was carried out in a general population
sample of over 4000 children aged 6-7 years in three London boroughs. Of the 2663 children
who donated shed teeth for analysis, 403 children were selected to form six groups, one each
of high (8 ug/g or more), intermediate (5-5.5 ug/g), and low (2.5 ug/g or less) tooth lead
levels for two socioeconomic groups (manual versus non-manual workers). Parents were inten-
sively interviewed at home regarding parental interest and attitudes toward education and
family characteristics and relationships. The early history of the child was then studied in
school using tests of intelligence (WISC-R), educational attainment, attention, and other cog-
nitive tasks. Teachers and parents completed the Conners behavior questionnaires. Results
showed that intelligence and other psychological measures were strongly related to social fac-
tors, especially social grouping. Lead level was linked to a variety of factors in the home,
especially the level of cleanliness and, to a lesser extent, maternal smoking. Before cor-
recting for confounding factors, there were significant associations between lead and full-
scale IQ scores; however, upon correcting for confounding factors, there were no statistically
significant associations between lead level and IQ or academic performance. Also, when rated
by teachers (but not by parents), there were small, reasonably consistent (but not statisti-
cally significant) tendencies for high-lead children to show more behavioral problems after
the different social covariables were taken into account statistically.
The Smith et al. (1983) study has much to recommend it: (1) a well-drawn sample of ade-
quate size; (2) three tooth lead groupings based on well-defined classifications minimizing
overlaps of exposure groupings based on whole tooth lead values, including quality-controlled
replicate analyses for the same tooth and duplicate analyses across multiple teeth from the
same child; (3) blood lead levels on a subset of 92 children which correlated reasonably well
with tooth lead levels (r = 0.45); (4) cross-stratified design of social groups; (5) extensive
information on social covariates and exposure sources; and (6) statistical control for poten-
tially confounding covariates in the analyses of study results. It should also be noted that
further statistical analyses of the Smith data, using tooth lead as a continuous variable or
finer-grain categorization of subjects into eight tooth lead exposure groups, have recently
been reported (Pocock and Ashby, 1985) to confirm no statistically significant associations
between tooth lead and IQ across the entire spectrum of lead exposure levels present among the
study population. Interestingly, the average full-scale IQ values for the medium- and high-
lead groups in the Smith study were 2 points below the average value for the control group.
Also, blood lead values for subsets of the children in the medium and high groups averaged
12-15 ug/dl (with all but one <30 ug/dl) upon sampling within a few months of neuropsychologic
testing around age six. Somewhat higher blood lead values may have been obtained if sampled
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at earlier ages for these children (given typical peaking of blood leads seen in preschool
children), but likely would have still fallen mainly in the 15-30 |jg/dl range.
Harvey et al. (1983, 1984) also recently reported on a study involving 189 children,
average age 2.5 years and 15.5 ug/dl blood lead, from the inner city of Birmingham, England.
The investigators utilized a wide range of psychometric tests, behavioral measures of activity
level, and psychomotor performance. They found that blood lead made no significant contribu-
tion to IQ decrements after appropriate allowance had been made for social factors, although,
consistent with findings from the Lansdown et al. (1986) study discussed below, a stronger
correlation between IQ and blood lead levels was found in children of manual workers (r =
-0.32) than in children of non-manual workers (r = +0.06). Strengths of this study are the
following: (1) a well-drawn sample; (2) extensive evaluation of 15 confounding social
factors; (3) a wide range of abilities evaluated; and (4) blind evaluations. The finding of
no significant associations between lead and IQ decrements at the relatively low blood levels
evaluated are consistent with the Smith study results discussed above for children in the same
exposure range.
Yule et al. (1981) carried out a pilot study on the effects of low-level lead exposure on
85 percent of a population of 195 children aged 6-12 years, whose blood lead concentrations
had been determined some nine months earlier as part of a European Economic Community survey.
The blood lead concentrations ranged from 7 to 32 ug/dl, and the children were assigned to
four quartiles encompassing the following values: 7-10 ug/dl; 11-12 ug/dl; 13-16 ug/dl; and
17-32 ug/dl. The tests of achievement and intelligence were similar to those used in the
Lansdown et al. (1974) and Needleman et al. (1979) studies. Significant associations were
reported between blood lead levels and decrements in IQ (full-scale IQ scores averaged ~7
points lower for the highest lead group), as well as lower scores on tests of reading and
spelling, but not mathematics (Yule et al., 1981). These differences in performance (although
reduced in magnitude) largely remained statistically significant at p <0.05 after age, sex,
and father's occupation were taken into account. However, other important potentially con-
founding social factors such as parental IQ were not controlled in this study, and the inves-
tigators cautioned against interpretation of their results as evidence of relationships
between lead and IQ or functioning at school without further confirmatory results obtained
after better control of social factors and other confounding variables.
Lansdown et al. (1986) replicated their earlier pilot study (Yule et al., 1981) with 194
children (X age = 8.8 yr) living in a predominantly working area of London near a busy road-
way. In this second, better designed study, a lengthy structured interview yielded data on
sources of exposure, medical history, and many potentially confounding variables, including
parental IQ and social factors. Analyses of covariance were used to evaluate the effects of
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lead and other factors on WISC-R verbal, performance, and full-scale IQ scores, as well as
reading accuracy and comprehension scores, for children with low (7-12 M9/dl) versus elevated
(13-24 ug/dl) blood lead levels. No significant effect of lead was evident even before con-
sidering social class. However, there was some suggestion of a trend in effects on IQ in the
manual working-class children when compared with non-manual working-class children.
In another study, Yule and Lansdown (1983) evaluated 302 children (X age = 9 yr) living
in Leeds, England. Tests and procedures similar to those employed in the previous two studies
were used and, in addition, a reaction time test was employed (Hunter et al., 1985). The
Leeds children were divided, for statistical analyses of the data, by (1) social class (manual
versus non-manual) and (2) blood lead level (low = 5-11 ug/dl; high = 12-26 M9/dl). As in the
London replication study, no statistically significant relationships for any of the IQ or
reading performance scores were found even before social class was controlled for in the sta-
tistical analyses. The high-lead children averaged essentially identical or very slightly
better than control subjects on several outcomes. On the other hand, small but statistically
significant (p <0.05) changes in reaction time (shorter for 3-sec delays; longer for 12-sec
delays) were found and appeared to parallel a similar pattern of reaction time effects of
larger magnitude reported by Needleman et al. (1979) for American children with higher lead
exposures. Analyses of covariance, controlling for age, revealed that the reaction-time dif-
ferences between low- and high-lead children in Leeds were only significant for the younger
children (aged 6-10 yr) but not for the older children (aged 11-14 yr).
Another paper by Yule et al. (1984) reported on the use of three different teacher ques-
tionnaires (Needleman, Rutter, and Conners) to assess attention deficits in the same children
evaluated in their earlier report (Yule et al., 1981). While there were few differences be-
tween groups on the Rutter Scale, the summed scores on the Needleman questionnaire across the
blood lead groupings approached significance (p = 0.096). Three of the questionnaire items
showed a significant dose-response function ("Day Dreamer," "Does not Follow Sequence of
Direction," "Low Overall Functioning"). Nine of 11 items were highly correlated with
children's IQ. Therefore, the Needleman questionnaire may be tapping IQ-related attention
deficits as opposed to measures of conduct disorder and socially maladaptive behavior (Yule et
al., 1984). The hyperactivity factors on the Conners and Rutter scales were reported to be
related to blood lead levels (7-12 versus 13-32 (jg/dl), but the authors noted that caution is
necessary in interpreting their findings in view of the crude measures of social factors
available and the differences between countries in diagnosing attention deficit disorders.
Moreover, since the blood lead values reported were determined only once (nine months before
psychological testing), earlier lead exposure may not be fully reflected in the reported blood
lead levels. However, even if somewhat higher earlier, it is likely that the blood leads
still mainly fell in the 15-30 |jg/dl range for the higher two quartile groups.
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Two recent reports by Schroeder and his colleagues (Schroeder et al., 1985; Schroeder and
Hawk, 1986) are of particular importance to the issue of lead's effects on children's cogni-
tive functioning. Although these studies dealt with children who had been identified through
lead-screening programs or who were potentially at risk for elevated lead exposure, the actual
blood lead levels measured in these children were, overall, in line with or not much higher
than the levels in the general population studies discussed above.
Schroeder et al. (1985) evaluated 104 lower SES children, ages 10 months to 6.5 years.
Approximately half of the children (age <30 months) were tested on the Bayley Scales of Mental
Development; the remainder of the subjects (age >30 months) were tested on the Stanford-Binet
Intelligence Scale. Several other variables were also assessed, including Caldwell and
Bradley (1979) HOME scores and parental IQ, SES, education, and employment. Venous blood
samples obtained on the day of testing were analyzed for lead concentrations and ranged from 6
to 59 pg/dl (X = 30 ug/dl). Statistical analysis of the data involved a form of hierarchical
backward stepwise regression. Lead was found to be a significant (p <0.01) source of the ef-
fect on IQ scores in these children after controlling for SES, HOME score, maternal IQ, and
other social factors. SES was the only other variable to reach statistical significance
(p <0.001); other variables apparently failed to reach significance because of collinearity
with SES. A corollary study of the same children by Milar et al. (1981a) found no association
between lead exposure and hyperactivity.
Fifty of the children were re-examined 5 years later, at which time all blood lead levels
were 30 ug/dl or lower. In addition to re-evaluating the children with the Stanford-Binet IQ
test, the investigators repeated SES and maternal IQ (but not HOME) measurements. Although
the 5-year follow-up IQ scores were negatively correlated with both contemporary and initial
blood lead levels, the effect of lead was not significant after covariates (especially SES)
were included in the regression model. It is interesting to note also that the correlation
between maternal and child IQ was only about 0.06 for children with initial blood lead levels
of 31-56 ug/dl, but returned to a nearly normal value of 0.45 after 5 years, when blood lead
levels had dropped. Similar findings have been reported by Perino and Ernhart (1974) and
Bellinger and Needleman (1983), and have been used to argue that an environmental factor
(i.e., lead) disrupts the normal mother-child IQ correlation of about 0.50. Thus, Schroeder
et al.'s (1985) finding provides further, indirect evidence of lead's disruptive effect on
children's cognitive functioning at blood lead levels in the range of approximately 30-60
ug/di.
Schroeder and Hawk (1986) replicated the above study with 75 Black children, all of low
SES and ranging in age from 3 to 7 years. Blood lead levels averaged 21 ug/dl (range: 6-47
|jg/dl). Backward stepwise multivariate regression analysis revealed a highly significant re-
lationship between contemporary blood lead level and IQ (p <0.0008); the effect was nearly as
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rtnhn, (p <0.002) whether
„ c,osest. SES was „„, . s,.gnificant
anaiyses sh°"ed HOME
or mean blood ,ead va,ues om
«'. a
°" IQ wwrrt to extend Hne.Hy across the entire range gf b]ooa
In fact, 78 percent of the subjects had blood ,ead ,.„„ be,ow 30
tu
00
oco
Otf)
120
110
100
90
80
70
60
50
I
•
J I
I
I I
10 15 20 25 30 35 40
BLOOD LEAD LEVEL.
46 50
Figure 12-2. Regression of IQ scores against blood lead levels, with 95%
confidence band. Double values indicated by triangle.
Source: Schroeder and Hawk (1986).
investigation that should be ^ed here has been reported by Silva et al
Thls preTiminary study investigated cognitive development and behavior problems in
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579 11-year-old children in Dunedin, New Zealand. Higher SES groups were significantly over-
represented in this sample, but the correlation between blood levels and SES was near zero.
The mean blood lead level at age 11 was 11.1 ug/dl (SD = 4.91). No significant effects on IQ
were evident from an analysis of WISC-R scores. Regression analyses and multiple correlations
were performed on scores from a reading ability test, the Rutter parent and teacher question-
naires, and other assessments of children's inattention and hyperactivity derived from parent
and teacher reports. The contribution of blood lead levels to the explained variance for the
reading ability scores was nonsignificant. However, five of the six remaining assessments of
children's behavior showed significant increases in the amount of explained variance when the
blood lead variable was added. Although blood lead accounted for only 0.8-1.2 percent of the
additional variance, the results nonetheless indicate some association between lead exposure
and small but significant adverse effects on behavior in older children, even after allowance
for certain background factors (e.g., maternal verbal ability, maternal depression, a com-
posite index of social disadvantage). A complementary report by Silva et al. (1986a) noted
that some of the children in the Dunedin pilot study had had significant exposure to lead
through paint-stripping activities in the home. Although only two subjects had blood lead
levels above 30 pg/dl at the time of testing, this backgorund information points up the need
for earlier and more precise characterization of long-term lead exposure for an accurate in-
terpretation of the Dunedin findings.
None of the general population studies reviewed here individually provide definitive evi-
dence for or against neuropsychologic deficits being associated with relatively low body lead
burdens in non-overtly lead-intoxicated children representative of general pediatric popula-
tions. The recent report by Schroeder and Hawk (1986) indicates a highly significant linear
relationship between a measure of IQ and blood lead levels over the range of 6 to 47 M9/dl.
This effect was almost equally as strong regardless of whether contemporary, past maximum, or
mean blood lead levels were used in the analysis. Because the subjects were all Black chil-
dren of uniformly low socioeconomic status, SES was not a significant covariate in the analy-
sis. On the other hand, this feature of the study limits the applicability of the findings to
the general U.S. population of children. It is possible that SES and lead exposure interact
such that IQ is affected by blood lead at lower SES levels but not at higher SES levels (cf.
Schroeder et al., 1985). Findings of stronger correlations between IQ and blood lead levels
in children of manual working class fathers (Harvey etal., 1983, 1984; Yule and Lansdown,
1983; Lansdown et al., 1986) are consistent with this supposition (cf. Winneke and Kraemer,
1984). If true, this interactive relationship would suggest that lower socioeconomic status
places children at greater risk to the deleterious effects of low-level lead exposure on
cognitive ability. However, as results from Schroeder et al. (1985) and Schroeder and Hawk
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(1986) indicate, other variables such as HOME scores and maternal IQ may covary with SES.
Other work (e.g., Milar et al., 1980; Dietrich et al., 1985b) points to the home environment
as a significant predictor of lead exposure. This close relationship between SES, quality of
home environment, and lead exposure suggests that SES may not be the sole determiner of in-
creased risk for cognitive impariment. Further research is needed to disentangle the relative
contributions of these variables to the neurotoxic effects of lead.
Of the other studies reviewed here, the Needleman analyses may be interpreted as provid-
ing acceptable evidence for full-scale IQ deficits of about 4 points and other neurobehavioral
deficits being associated with lead exposures of American children resulting in dentine lead
values that exceed 20-30 ppm and likely average blood lead values in the 30-50 ug/dl range.
The report of recent analyses by Schroeder et al. (1985) supports this conclusion, even after
the major influence of SES was allowed for in the analyses. However, their findings indicate
that the effect of blood lead on IQ could not be detected five years after the original as-
sessment. A follow-up by Bellinger et al. (1984b) of the children studied by Needleman et al.
(1979) suggests that other measures of classroom performance may show long-term effects of
early lead exposure more effectively than IQ measures (see also Silva et al., 1986b). Shaheen
(1984) has also questioned the sensitivity of IQ scores and has suggested that the variability
in outcomes of studies of lead's effects on neuropsychological functioning in children may
originate with differences in the ages at which children are subjected to toxic lead expo-
sures.
For the most part, the remaining general population studies reviewed in this section
report a lack (with a few exceptions) of statistically significant effects on IQ or other
neuropsychologic measures. Most of the remaining studies found slightly lower IQ scores for
higher-lead exposure groups than for low-lead control groups before correcting for confounding
variables, but the differences were typically reduced to 1-2 IQ points and were non-signifi-
cant (usually even at p <0.10) upon correction for confounding factors. The following con-
clusions may be stated about these latter results: (1) they are suggestive of relatively
minimal (if any) effects of lead on IQ in general populations, especially in comparison to the
much larger effects of other factors (e.g., social variables), at the exposure levels evalu-
ated in these studies (blood lead values mainly in the 15-30 |jg/dl range); and (2) they are
not incompatible with findings of significant lead effects on IQ at higher average blood lead
levels (£30 ug/dl).
The few exceptions to the general pattern noted above warrant comment here. The pilot
study by Yule et al. (1981, 1984), which found significant (6-7 point) IQ decrements and
poorer ratings on several categories of classroom behavior, has certain methodological limi-
tations; specifically, the study provided only relatively crude control for socioeconomic
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factors (as noted by the authors) and it failed to take parental IQ into account at all. In
comparison to other studies, the reported IQ decrements of about 6-7 points are consistent
with neither (1) the maximum 1-2 point IQ differences seen in other general population studies
of children with comparable lead exposures (mainly in the 15-30 (jg/dl blood lead range), nor
(2) the results of clinic studies showing 4-5 point IQ decrements at distinctly higher lead
levels (i.e., at >30 ng/dl). However, the findings of altered reaction time patterns by
Hunter et al. (1985), which parallel those reported by Needleman at higher exposure levels,
are somewhat more credible and appear to argue for probable effects of lead on attention or
vigilance functions at levels extending below 30 pg/dl and, possibly, down to as low as
15-20 pg/dl.
12.4.2.2.2.3 Smelter area studies. The smelter studies evaluated children with elevated
lead exposures associated with residence in cities or elsewhere in close proximity to lead-
emitting smelters. Most of the early studies, conducted in the 1970s, found mixed results
even though evaluating children with blood lead levels typically in excess of 30 ug/dl. Be-
cause of methodological weaknesses, however, virtually all of the early studies must be viewed
as inconclusive.
For example, in an early study of this type Lansdown et al. (1974) reported a relation-
ship between blood lead level in children and the distance they lived from lead-processing
facilities, but no relationship between blood lead level and mental functioning. However,
only a minority of the lead-exposed cohort had blood lead levels markedly differing from
control subjects with elevated blood lead levels (<40 ug/dl). Furthermore, this study failed
to adequately consider important confounding factors such as socioeconomic status.
In another study, Landrigan et al. (1975) found that lead-exposed children living near an
El Paso, Texas, smelter scored significantly lower than matched controls on measures of per-
formance IQ and finger-wrist tapping. The control children in this study were, however, not
well matched by age or sex to the lead-exposed group, although the results remained statisti-
cally significant after adjustments were made for age differences. In contrast, McNeil and
Ptasnik (1975) found little evidence of lead-associated decrements in cognitive abilities in
another sample of children living near the same lead smelter in El Paso. These children who
were generally comparable medically and psychologically to matched controls living elsewhere
in the same city except for the direct effects of lead (blood lead level, free erythrocyte
protoporphyrin levels, and X-ray findings). An extensive critique of these two El Paso
studies was performed by another expert committee (see Muir, 1975), which concluded that no
reliable conclusions could be drawn from either of the published studies in view of various
methodological and other problems affecting their conduct and statistical analyses.
A later study by Ratcliffe (1977) of children living near a battery factory in Manches-
ter, England, found no significant associations between blood lead levels sampled at two years
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of age (28 ug/dl versus 44 ug/dl in low- versus high-lead groups) and testing done at age five
on the Griffiths Mental Development Scales, the Frostig Developmental Test of Visual Percep-
tion, a pegboard test, or a behavioral questionnaire. The differences in scores, although
small, were somewhat better for the low-lead exposure children than for the higher exposure
group. The small sample size (23 low-lead and 24 high-lead children), inadequate control for
parental IQ, and the failure to repeat blood lead assays at age five weaken this study.
Variations in blood lead levels occurring after age two among control children may have less-
ened exposure differences between the low- and high-lead groups, and larger sample sizes would
have better allowed for detection of any lead effects present.
The more recent smelter studies, described next, provide assessments that generally ac-
cord somewhat greater attention to control for potentially confounding factors. Also, some of
the studies assessed larger samples of children, presumably allowing more accurate estimation
of any lead effects present.
Two studies by Winneke and colleagues, the first a pilot study (Winneke et al., 1982a)
and the second an extended study (Winneke et al., 1983), employed tooth lead analyses anal-
ogous to some of the studies already discussed above. In the pilot study, incisor teeth were
donated by 458 children aged 7-10 years in Duisburg, Germany, an industrial city with airborne
lead concentrations between 1.5 and 2.0 (jg/m3. Two extreme exposure groups were formed, a
low-lead group with 2.4 ug/g mean tooth lead level (n = 26) and another, high-lead group with
7 ug/g mean tooth lead level (n = 16). These groups were matched for age, sex, and father's
occupational status. The two groups did not differ significantly on confounding covariates,
except that the high-lead group showed more perinatal risk factors. Parental IQ and quality
of the home environment were not among the 52 covariables examined. The authors found a mar-
ginally significant decrease (p <0.10) of 5-7 IQ points and a significant decrease in percep-
tual-motor integration (p <0.05), but no significant differences in hyperactivity as measured
by the Conners Teachers' Questionnaire administered during testing. As with the Yule et al.
(1981) study, the inadequacy of statistical or other control for background social variables
and parental IQ (as well as group differences in perinatal factors) weaken this study; the in-
vestigators cautioned against interpretation of their results as evidence for low-level lead
exposure effects in the absence of further, confirmatory results from larger, better con-
trolled studies (such as those conducted by them elsewhere as described below).
In their second study, Winneke et al. (1983) evaluated 115 children (X age =9.4 years)
living in the lead smelter town of Stolberg, Germany. Tooth lead (X = 6.16 ppm, range =
2.0-38.5 ppm) and blood lead levels (X = 13.4 ug/dl; range = 6.8-33.8 ug/dl) were signifi-
cantly correlated (r = 0.47; p <0.001) for the children studied. Using stepwise multiple
regression analysis, the authors found significant (p <0.05) or marginally significant (p
<0.10) associations between tooth lead levels and measures of perceptual-motor integration,
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reaction-time performance, and four behavioral rating dimensions, including distract!bility.
This was true even after taking into account age, sex, duration of labor at birth, and
socio-heredity background as covariates. However, the proportion of explained variance due to
lead never exceeded 6 percent for any of these outcomes, and no significant association was
found between tooth lead and WISC verbal IQ after the effects of socio-hereditary background
were eliminated.
A third study by Winneke et al. (1984) evaluated neuropsychologic functioning and neuro-
physiological parameters for 122 children (aged 6-7 yr) living in the Nordenham, FRG area.
Performance on a variety of neuropsychologic tests (shortened form of the Hamburg-Wechsler IQ
test; reaction-behavior and reaction-time tests, etc.) was evaluated in relation to both con-
currently sampled blood lead values (X = 8 ug/dl; max. = 23 ug/dl) and umbilical cord blood
lead levels (max. = 31 ug/dl). A variety of potentially confounding factors (such as socio-
hereditary variables, pre- and postnatal risk factors, etc.) were also assessed and taken into
account in a series of stepwise multiple regression analyses in which the effects of confound-
ing factors were successively eliminated and the effects of lead then checked for signifi-
cance. No significant associations (at p <0.05) were found between either umbilical cord or
current blood lead levels and verbal, performance, or total IQ scores estimated from the
Hamburg-Wechsler subtests (only the correlation for performance IQ with current blood lead
level reached p <0.10). On the other hand, much larger and highly significant correlations
were found between socio-hereditary factors and all three types of IQ scores. The investi-
gators remarked on the heavy dependence of the IQ measurements on the social environment and
noted that, as in their prior large-scale study (Winneke et al., 1983), it was not possible to
convincingly show a lead-dependent decrease in intelligence. Nor were any lead effects found
on the Goettinger shape reproduction test of psychomotor performance or for various reaction-
time measures. Only in the case of reaction behavior, as indexed by increased errors on the
Wiener (Vienna) serial stimulus reaction test, were significant deficits in neuropsychological
functioning detected at the low exposure levels (<25-30 ug/dl) evaluated in this study. Cer-
tain statistically significant effects on electrophysiological measures of neurophysiological
functioning were also observed (as described below in Section 12.4.2.2.2.7).
The above smelter area studies generally do not provide much evidence for cognitive or
behavioral deficits being associated with lead exposure in non-overtly lead exposed children,
except perphaps for the reaction-behavior deficits reported by Winneke et al. (1984). The
lack of convincing evidence for IQ deficits at the blood lead levels (generally 15-30 ug/dl)
typifying the pediatric populations studied by Winneke comport well with the same type of
findings reported by British investigators (Yule; Smith; Harvey) for general population groups
with similar lead exposure ranges. At the same time, the possibility of small neuropsy-
chologic deficits being associated with lead exposure in apparently asymptomatic children at
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the exposure levels studied cannot be completely ruled out, given the overall pattern of re-
sults obtained with the cross sectional study designs employed by Winneke and the British in-
vestigators. Small, 1-2 point differences in IQ seen in some of their studies between control
and lead exposure groups might in fact be due to lead effects masked by much larger effects of
socioeconomic factors, home environment, or parental IQ. At the same time, the very small or
nil differences in IQ seen in these studies for children with blood lead levels mainly in the
15-30 ug/dl range suggest that, if the IQ decrements are in fact due to lead, then it is ex-
tremely unlikely that any IQ effects (of presumably even smaller magnitude) would be convinc-
ingly detectable at lower blood lead levels.
12.4.2.2.2.4 Studies of neuropsychiatrically disordered children. Rather than starting
with a known lead-exposed population and attempting to discover evidence of neurobehavioral
dysfunction, a number of studies have first identified a population with some recognized dis-
order and then looked for evidence of elevated lead exposure. For example, a series of
studies by David et al. (1972; 1976a,b; 1977; 1979a,b; 1982a,b; 1983; 1985) measured lead
levels in diagnosed hyperkinetic children and showed an association between hyperactivity and
elevated lead levels. However, whether a disorder such as hyperactivity is the effect or the
cause of elevated lead exposure is a difficult issue to resolve. It is possible, for example,
that hyperactive children might ingest more lead than normal children because of a greater
incidence of pica or even because they stir up more dust-borne lead by their activity. How-
ever, David et al. (1977) reported that blood lead levels of hyperactive children with a
probable etiology of an organic nature were lower than those of children with no apparent
cause (other than lead). This finding suggests that hyperactivity does not necessarily result
in elevated lead exposure, but it does not rule out the possibility of a third factor causing
both hyperactivity and elevated blood lead levels (see discussion of Gittelman and Eskenazi,
1983, below). Also, a problem common to the studies in question is the lack of adequate in-
formation on the children's past exposure to lead, particularly during preschool years when
children tend to be at greatest risk to higher exposure levels. As David et al. (1976a) have
acknowledged, it is difficult to establish an etiological relationship between lead and
behavioral disorders on the basis of retrospective estimations of lead exposure.
A recent study by David et al. (1983) appeared to obviate some of the problems of the
correlational approach by experimentally manipulating body lead levels, i.e., by reducing
blood lead concentrations through the administration of a chelating agent, penicillamine. The
objective was to determine if decreases in body lead would be accompanied by improvements in
children's hyperactive behavior, and in short, this was essentially the conclusion drawn by
David and his colleagues. In addition, the study compared the effect of the chelating agent
with a therapeutic drug of known efficacy, methylphenidate, and found the two treatments to be
roughly equivalent in reducing symptoms of hyperactivity.
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Although this study by David et al. (1983) was in many respects well designed and exe-
cuted, certain problems nevertheless cloud its interpretation. As noted by Needleman and
Bellinger (1984), the number of subjects per treatment group was rather limited (maximum of
31) and quite unbalanced due in part to a high and disproportionate subject attrition rate.
Subjects were particularly prone to drop out of the placebo group, and this imbalance was
exacerbated by a "chance preponderance" of subjects assigned to the penicillamine treatment
and by later reassignment of some placebo and methylphenidate subjects to the penicillamine
group. Questions might also be raised concerning the appropriateness of the statistical
treatment of data by David et al. (1983). For example, multivariate analysis of variance
(MANOVA) would seem to be more appropriate than separate ANOVAs and multiple t-tests applied
to the various outcome measures used to assess the children's behavior. Use of MANOVA would
also have helped alleviate the problem of regression toward the mean, which in this case may
have created the false impression that "improvements" in behavior, i.e., changes toward more
normal behavior, were due to an effect of the treatment. Rutter (1983, p. 313) has also noted
that David's multiple group comparisons are not as convincing as an analysis that would util-
ize individual blood lead and behavior scores (presumably, multivariate regression analysis).
Finally, as David et al. (1983) themselves point out, it is clear that lead could be only one
of several etiological factors in the causation of hyperkinesis or attention deficit disorders
in children and that, at best, their findings pertain only to recognized hyperactive children,
not to the general population.
An attempt by Gittelman and Eskenazi (1983) to replicate earlier work by David et al.
(1972; 1977) was only partly supportive of the letter's findings. A large group of hyper-
active children (n = 103) showed a trend (p = 0.06) toward higher chelated lead levels in
their urine, but a clear-cut (p = 0.02) elevation in lead levels was evident only in paired
comparisons with 33 nonhyperkinetic siblings. As Gittelman and Eskenazi (1983) noted, this
finding raises the question of why the hyperactive children had higher lead levels than their
siblings, given that they shared the same water, air, and home environment. The possibility
of a third factor, e. g., a metabolic difference that might affect the ability to excrete lead
as well as the occurrence of hyperactivity, cannot be dismissed.
A study of 98 Swedish children with various minor neuropsychiatric disorders (e.g., per-
ceptual-motor dysfunctions, speech disorders, attention deficit problems) found no correlation
between the children's disorders and their tooth lead levels (Gillberg et al., 1982). How-
ever, comparing the 10 highest and 10 lowest lead-burdened children did reveal a significant
difference in a clinical measure of their mean reaction times.
Youroukos et al. (1978) compared the blood lead as well as ALA-D values of 60 Greek
children with mental retardation of unknown etiology against 30 mentally retarded children
with a known etiology and 30 normal children. The average values of the mentally retarded
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patients were significantly different from both of the control groups in two regards: blood
lead "level was higher (30 pg/dl versus 21 (jg/dl in both control groups) and, in 14 patients
with elevated U 40 ug/dl) blood lead levels, ALA-D activity was significantly lower.
Although pica was noted to be common in both groups of mentally retarded children, no child in
the study was known to have ever been lead-poisoned.
Work in Scotland has provided information tending to link prenatal lead exposures to the
later development of mental retardation. Beattie et al. (1975) identified 77 retarded chil-
dren and 77 normal children matched on age, sex, and geography. The residence during the ges-
tation of the subject was determined, and a first-flush morning sample of tap water was ob-
tained from the residence. Of 64 matched pairs, no normal children were found to come from
homes served with water containing high lead levels (>800 ug/liter), whereas 11 of the 64 re-
tarded children came from homes served with such high-lead water. The authors concluded that
pregnancy in a home with high lead in the water supply increases by a factor of 1.7 the risk
of bearing a retarded child. In follow-up work, Moore et al. (1977) obtained lead values from
blood samples drawn during the second week of life from children studied by Beattie et al.
The samples had been obtained as part of routine screening for phenylketonuria and kept stored
on filter paper. Blood samples were available for 41 of the retarded and 36 of the normal
children in the original study by Beattie et al. Blood lead concentrations in the retarded
children were significantly higher than values measured in normal children: the mean for
retardates was 1.23 ± 0.43 umol/liter (25.5 ±8.9 ug/dl) and for normals was 1.0 ± 0.38
umol/liter (20.9 ± 7.9 ug/dl). The difference in lead concentrations was significant (p =
0.02) by the Mann-Whitney test.
These latter two studies suggest that lead exposure to the fetus during the critical
period of brain development may cause perturbations in brain organization that are expressed
later in mental retardation syndromes, and they raise for careful scrutiny the issue of post-
natal risks associated with intrauterine exposure to lead. Long-term prospective studies of
the type described next are beginning to produce results which address that issue.
12.4.2.2.2.5 Prospective Studies of Neurobehavioral Effects of In-Utero or Early Post-
natal Lead Exposures. During recent years a number of prospective studies have been initiated
in the United States and abroad (Europe, Australia, etc.). These studies emphasize the
following: (1) the documentation of lead exposure histories during pregnancy, at birth, and/
or postnatally well into later years of childhood; and (2) the evaluation of relationships
between such lead exposures and delays in early postnatal physical or neurological development
and, also, subsequent alterations in normal neuropsychological and neurophysiological func-
tions. Progress in a number of these studies was discussed at the Second International Con-
ference on Prospective Lead Studies held in April, 1984 (Bornschein and Rabinowitz, 1985).
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Initial results have been obtained from two of these studies and are of particular interest
here.
As part of a longitudinal study of early developmental effects of lead, Bellinger et al.
(1984a) administered Bayley Scales of Infant Development at age 6 months to infants born at a
Boston hospital. The infants were classified into three groups according to umbilical cord
blood lead levels obtained at birth: low (X = 1.8 u/dl); middle (X = 6.5 M9/dl); and high (X =
14.6 ug/dl; none exceeded 30 ug/dl). Multiple regression analyses indicated that the "high"
cord blood-lead levels were significantly associated with lower covariance-adjusted scores on
the Bayley Mental Development Index, but scores on the Psychomotor Development Index were not
related to cord blood-lead levels. Infant blood-lead levels sampled at 6 months of age were
not associated with scores on either the Mental or Psychomotor Development Index. These data
were interpreted by Bellinger et al. (1984a, 1985) as being compatible with the hypothesis
that low levels of lead delivered transplacentally to the fetus are toxic to the newborn
infant. However, although the results suggest that jrn utero exposure may result in delays in
early development during the first 6 months postnatally, the results do not allow estimation
of the persistence of the observed delays in postnatal neurobehavioral development.
Dietrich et al. (1985a) also recently reported initial results emerging from a long-term
prospective study of infants born in Cincinnati, Ohio. The Bayley Mental Development Index
(MDI), Psychomotor Development Scale (PDS), and Infant Behavior Record (IBR) were administered
at 3, 6, 12, and 24 months to infants not born at significant biological risk due to non-lead
factors (such as low birth weights, etc.). The Home Observation for Measurement of the Envi-
ronment (HOME) scales (Caldwell and Bradley, 1979) were used to assess and statistically con-
trol for relevant factors in the rearing environments of the infants, and blood samples were
obtained at birth (umbilical cord blood), 10 days, and every three months thereafter. Geo-
metric mean blood lead levels increased from 6.11 ug/dl at 3 months to 14.87 ug/dl at 12
months and included maximum values of 28, 33, 55, and 46 ug/dl at the 3, 6, 9 and 12 month
sampling points. Based on regression analyses between blood lead values at those time points
and MDI scores at 3, 6, and 12 months, only an unadjusted negative lag correlation between
blood levels at 3 months and MDI at 6 months was significant; but that correlation was sub-
stantially reduced and no longer significant after adjustment for HOME scores. Free erythro-
cyte protoporphyrin levels at 6 months were significantly correlated to 6-month MDI scores and
remained so after correction for HOME scores. As for IBR data, only "Sensory Interest" at
12 months was significantly negatively correlated with 6 or 12 month blood lead levels (at
p <0.05 and p <0.01, respectively). The lag correlation between 6 month blood leads and 12
month IBR "Sensory Interest" was not significant after adjustment for HOME scores, but the
correlation with 12 month blood leads remained significant after adjustment for HOME scores.
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The investigators concluded, based on these initial results, that low to moderate lead expo-
sure during the first year of life has only a small impact (if any) on early sensorimotor
development.
(More recent significant results from these other longitudinal studies are reviewed and
assessed in the Addendum to this document.)
12.4.2.2.2.6 Studies of association of neuropsychologic effects and hair lead levels.
Several studies have reported significant associations between hair lead levels and behavioral
or cognitive testing endpoints (Pihl and Parkes, 1977; Hole et al., 1979; Hansen et al., 1980;
Capel et al., 1981; Ely et al., 1981; Thatcher et al., 1982; Marlowe et al., 1982, 1983, 1985;
Marlowe and Errera, 1982). Measures of hair lead are easily contaminated by external expo-
sure and are generally questionable in terms of accurately reflecting internal body burdens
(see Chapter 9). Such data, therefore, cannot be credibly used to evaluate relationships
between absorbed lead and nervous system effects and are not discussed further here.
12.4.2.2.2.7 Electrophysiological studies of lead effects in children. In addition to
psychometric and behavioral approaches, electrophysiological studies of lead neurotoxicity in
non-overtly lead-intoxicated children have been conducted. One such study (Thatcher et al. ,
1984) reported significant effects on various measures of auditory and visual evoked poten-
tials in lead-exposed children, but the only measure of lead exposure was hair lead, which,
as previously noted, is not a suitable index of lead exposure.
Burchfiel et al. (1980) used computer-assisted spectral analysis of a standard EEC exam-
ination on 41 children from the Needleman et al. (1979) study and reported significant EEC
spectrum differences in percentages of alpha and low-frequency delta activity in spontaneous
EEGs of the high-lead children. Percentages of alpha and delta frequency EEG activity and
results for several psychometric and behavioral testing variables (e.g., WISC-R full-scale IQ
and verbal IQ, reaction time under varying delay, etc.) for the same children were then
employed as input variables (or "features") in direct and stepwise discriminant analyses. The
separation determined by these analyses for combined psychological and EEG variables
(p <0.005) was reported to be strikingly better than the separation of low-lead from high-lead
children using either psychological (p <0.041) or EEG (p <0.079) variables alone. Unfortun-
ately, no dentine lead or blood lead values were reported for the specific children from the
Needleman et al. (1979) study who underwent the EEG evaluations reported by Burchfiel et al.
(1980). Lead-exposure levels associated with the observed EEG effects would appear likely to
fall within the same broad 30-50 ng/dl blood lead range estimated earlier for the Needleman IQ
deficit observations.
Guerit et al. (1981) examined 79 11-year-old children attending three different schools
in the vicinity of a lead smelter and presenting blood lead levels up to 44 |jg/dl (averaging
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less than 30 ug/dl). Children from two distant urban and rural schools served as controls. A
neurophysiological function score for each child was based on measures of EEGs, visual evoked
potentials, brainstem auditory evoked potentials, and eye movements. Neurophysiological
scores were negatively correlated (p <0.05 by Spearman rank correlation coefficient) with
blood lead and FEP levels for the children from one of the smelter area schools, but the
authors attributed this finding to the inclusion of four children who were left-handed or
suffering from external ear pathology. Chi-square tests of neurophysiological scores as a
function of blood lead or FEP groupings based on the total study population were all non-
significant. Note that comparatively low power nonparametric statistical tests were employed
in this study because of the qualitative or ordinal nature of the data. However, the use of
more detailed quantitative measures of neurophysiological function would have enabled the in-
vestigators to employ more powerful parametric statistics, with possibly different outcomes
from their analyses.
The relationship between low-level lead exposure and neurobehavioral function (including
electrophysiological responses) in children aged 13-75 months was extensively explored in
another study, conducted at the University of North Carolina in collaboration with the U.S.
Environmental Protection Agency. Psychometric evaluation revealed a significant lead-related
IQ decrement at the time of initial evaluation (Schroeder et al., 1985), as noted previously.
No relationship between blood lead and hyperactive behavior (as indexed by standardized play-
room measures and parent-teacher rating scales) was observed in these children (Milar et al.,
1981a). On the other hand, electrophysiological assessments, including analyses of slow cort-
ical potentials during sensory conditioning (Otto et al., 1981) and EEG spectra (Benignus et
al., 1981), did provide evidence of CNS effects of lead in the same children. A significant
linear relationship between blood lead (ranging from 6 to 59 ug/dl) and slow wave voltage
during conditioning trials was observed (Otto et al., 1981), as depicted in Figure 12-3.
Analyses of quadratic and cubic trends, moreover, did not reveal any evidence of a threshold
for this effect. The slope of the blood lead x slow wave voltage function, however, varied
systematically with age. No effect of blood lead on EEG power spectra or coherence measures
was observed, but the relative amplitude of synchronized EEG between left and right hemis-
pheres (gain spectra) increased relative to blood lead levels (Benignus et al., 1981). A
significant cubic trend for gain between the left and right parietal lobes was found with a
major inflection point at 15 ug/dl. This finding suggests that EEG gain is altered at blood
lead levels as low as 15 ug/dl, although the clinical and functional significance of this mea-
sure has not been established.
A follow-up study of slow cortical potentials and EEG spectra in a subset (28 children
aged 35-93 months) of the original sample was carried out two years later (Otto et al., 1982).
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(9
UJ
I
30
20
10
0
-10
-20
-30
-40
10
0
-10
-20
-30
20
10
0
-10
-20
MONTHS
I I 1 I I 1 I
(b)
AGE. months
• 1223
• 2435
A 3647
I
I
I
\ I
1 I
\ I
(el'
o 48 59
O 6075
I I I
I
J_
5 10 15 20 25 30 35 40 45 50 55
PbB LEVEL, pg/dl
Figure 12-3. (a) Predicted slow wave voltage and 95% confidence
bounds in 13- and 75-month-old children as a function of blood
lead level, (b) Scatter plots of slow wave data from children aged
13 to 47 months with predicted regression lines for ages 18, 30,
and 42 months, (c) Scatter plots for children aged 48 to 75
months with predicted regression lines for ages 54 and 66 months.
These graphs depict the linear interaction of blood lead and age.
Source: Ottoetal. (1981).
12-105
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Slow wave voltage during sensory conditioning again varied as a linear function of blood lead,
even though the mean lead level had declined by 11 ug/dl (from 32.5 ug/dl to 21.1 ug/dl).
Although the EEG gain effect did not persist, the similarity of slow wave voltage results ob-
tained at initial and follow-up assessments suggests that the observed alterations in this
parameter of CNS function were persistent, despite a significant decrease in the mean blood
lead level during the two-year interval.
In a five-year follow-up study on a subset of the same children, Otto et al. (1985) found
that slow wave voltage varied as a function of current blood lead level during active condi-
tioning, but not during the passive conditioning test used in earlier studies. In the passive
test, a tone was paired with a short blackout of a silent cartoon. The active test was simi-
lar except that children pressed a button to terminate the blackout and resume the cartoon.
Although the brain response elicited by the active test is greater than that produced by the
passive test, the active test cannot be performed reliably by children under five years of
age.
In addition to the experimental conditioning tests, Otto et al. (1985) used two clini-
cally validated measures of sensory function, the pattern-reversal visual evoked potential
(PREP) and the brainstem auditory evoked potential (BAEP). Exploratory analysis of PREPs
revealed increased amplitude and decreased latency of certain components as a linear function
of original blood lead levels. Although these results were contrary to predictions, the find-
ings are consistent with the results of Winneke et al. (1984), who found an association
between increased blood lead level and decreased latency in the primary positive component of
PREPs in children. BAEP results of the five-year follow-up study also indicated significant
associations between original blood lead levels and increased latencies of two components
(waves III and V), indicative of auditory nerve conduction slowing.
Otto and his coworkers (Otto, 1986; Robinson et al., 1985) recently reported the results
of a replication study with an independent group of children 3 to 7 years old. Blood lead
levels ranged from 6 to 47 ug/dl at the time of testing. Psychometric data from this study
(Schroeder and Hawk, 1986) have been reviewed above. Sensory conditioning was limited to the
passive test due to the age range of the children. Contrary to earlier findings (Otto et al.,
1981, 1982), slow wave voltage did not vary with blood lead levels. Differences between the
two groups studied, however, may have contributed to the discordant results. Children in the
earlier studies were somewhat younger (1-6 versus 3-7 years) and were exposed by different
routes (secondary occupational exposure versus lead paint and contaminated soil) than children
in the replication study (see review by Otto, 1986). Until further studies are undertaken to
clarify the inconsistent slow wave results, earlier findings must be interpreted cautiously.
12-106
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Inconsistencies in PREP and BAEP results between the five-year follow-up and replication
studies were also found. Only one PREP amplitude measure varied systematically with blood
lead levels in the replication study, and this was in the opposite direction from previous
findings. BAEP results of the replication study were considerably more complex and only par-
tially consistent with the five-year follow-up study. Several BAEP latency measures showed a
curvilinear relationship to maximal blood lead levels, whereas a simple linear relationship
was observed in the earlier study. That is, BAEP latencies in the replication study decreased
as blood lead levels rose from 6 to 25 ug/dl, but increased at higher PbB levels. The des-
cending limb of this curve paralleled the findings of Winneke et al. (1984), who observed
faster peripheral nerve conduction velocities as well as decreasing latency in the primary
positive PREP component of children with blood lead levels up to 23 ug/dl. On the other hand,
the ascending limb of the BAEP latency curve was consistent with the five-year follow-up
results. Moreover, the I-V interpeak latency, a measure of central transmission time in the
auditory pathway, increased linearly with increasing blood lead levels in the replication
study. In addition, hearing threshold, a reflection of peripheral auditory system function,
increased directly with lead levels. Although hearing threshold did not vary with blood lead
level in the five-year follow-up study (Otto et al., 1985), this finding bears further in-
vestigation in view of other reports suggesting impaired auditory processing in lead-exposed
children (de la Burde and Choate, 1975; Needleman et al., 1979).
In summary, these electrophysiological studies provide suggestive evidence of lead-
related effects on CMS function in children at blood lead levels considerably below 30 ug/dl,
but inconsistent findings across studies require clarification. Linear dose-response rela-
tions have been observed in slow-wave voltage during conditioning (Otto et al., 1981, 1982,
1985), BAEP latency (Otto et al., 1985), PREP latency (Otto et al., 1985; Winneke et al.,
1984), and PREP amplitude (Otto, 1986; Otto et aT., 1985a), although the specific components
affected and direction of effect varied across studies. Sensory evoked potentials, in parti-
cular, hold considerable promise as sensitive, clinically valid nervous system measures un-
affected by social factors that tend to confound traditional psychometric measures (Halliday
and McDonald, 1981; Prasher et al., 1981). BAEPs, for instance, are not altered by changes in
attention or level of consciousness. Reliable BAEPs can be recorded in (sedated) children
between the ages of one and five, the most vulnerable period for lead poisoning as well as the
most difficult period for most types of neurobehavioral testing. The current electrophysio-
logical evidence concerning lead exposure and brain function in children, however, is too
fragmentary to draw any firm conclusions. The use of evoked potential measures in prospective
pediatric lead studies would provide a very useful adjunct to other neurobehavioral tests and
would help to resolve current uncertainties regarding the neurobehavioral threshold of lead
toxicity.
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The adverse effects of lead on peripheral nerve function in children remain to be con-
sidered. Lead-induced peripheral neuropathies, although often seen in adults after prolonged
exposures, are rare in children. Several articles (Anku and Harris, 1974; Erenberg et al.,
1974; Seto and Freeman, 1964), however, describe case histories of children with lead-induced
peripheral neuropathies, as indexed by electromyography, assessment of nerve conduction veloc-
ity, and observation of other overt neurological signs, such as tremor and wrist or foot
drop. Frank neuropathic effects have been observed at blood lead levels of 60-80 pg/dl
(Erenberg et al., 1974). In one case study (Seto and Freeman, 1964), signs indicative of
peripheral neuropathy were reported to be associated with blood lead values of 30 ug/dl; how-
ever, lead lines in long bones suggested probable past exposures leading to peak blood lead
levels at least as high as 40-60 ug/dl and probably in excess of 60 (jg/dl (based on the data
of Betts et al., 1973). In all of these case studies, some, if not complete, recovery of
affected motor functions was reported after treatment for lead poisoning. A tentative associ-
ation has also been hypothesized between sickle cell disease and increased risk of peripheral
neuropathy as a consequence of childhood lead exposure. Half of the cases reported (10 out of
20) involved inner-city Black children, several with sickle cell anemia (Anku and Harris,
1974; Lampert and Schochet, 1968; Seto and Freeman, 1964; Imbus et al., 1978). In summary,
evidence exists for frank peripheral neuropathy in children, and such neuropathy can be
associated with blood lead levels at least as low as 60 ug/dl and, possibly, as low as 40-60
ug/dl.
Further evidence for lead-induced peripheral nerve dysfunction in children is provided by
two studies by Feldman et al. (1973a,b, 1977) of inner city children and from a study by
Landrigan et al. (1976) of children living in close proximity to a smelter in Idaho. The
nerve conduction velocity (NCV) results from the latter study are presented in Figure 12-4 in
the form of a scatter diagram relating peroneal nerve conduction velocities to blood lead
levels. No clearly abnormal conduction velocities were observed, although a statistically
significant negative correlation was found between peroneal NCV and blood lead levels (r =
-0.38, p <0.02 by one-tailed t-test). These results, therefore, provide evidence for signi-
ficant slowing of nerve conduction velocity (and, presumably, for advancing peripheral neuro-
pathy as a function of increased blood lead levels), but do not allow clear statements re-
garding a threshold for pathologic slowing of NCV.
In a recent study mentioned earlier, Winneke et al. (1984) evaluated neurophysiological
functions as well as neuropsychologic performance in children from Nordenham, FRG. Results
from a standard neurological examination and sensory nerve conduction velocities of the radial
and median nerves were analyzed in relation to concurrent blood lead values and umbilical cord
blood lead levels sampled approximately six years earlier. Contrary to expectations, in-
creasing conduction velocities for radial and median nerves were found to be significantly
12-108
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!
88.00
82 90
77 GO
72.40
t 6720
8
o
6200
Q 56.80
51.60
46.<
41.20
36.00
~~| 1 1 1 1 1 I
Y (CONDUCTION VELOCITY) = 54.8 - .045 x (BLOOD LEAD)
(r = -0.38) (n = 202)
• • * • M* * • •*
%7JJ •*w^** •** *
i i
i
0 15 30 45 60 75 90 105 120 135 150
BLOOD LEAD. M9/dl
Figure 12-4. Peroneal nerve conduction velocity versus blood lead level, Idaho,
1974.
Source: Landrigan et al (1976).
12-109
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associated with current blood lead levels (at p <0.01 and <0.10, respectively). As noted
above, visual evoked potentials showed a significantly decreased latency in one component,
which suggested more rapid conduction in the visual pathway, consistent with the peripheral
nerve conduction findings. Somatosensory evoked potentials showed no significant effect; nor
were associations found between any of the electrophysiological measures and cord blood lead
levels or any of a number of socio-hereditary background variables (the latter of which were
strongly related to neuropsychologic outcome results).
The lead-associated increases in nerve conduction observed by Winneke et al. (1984) for
children with blood lead levels below 25-30 ug/dl differ from previously noted findings of
slowed NCVs being associated with increasing blood lead values above 30 ug/dl. However, the
apparently paradoxical findings were noted by the investigators as being consistent with those
of Englert (1978), who similarly found an increase in the motor NCV of the median nerve among
lead-exposed children in Nordenham. Winneke et al. (1984) nevertheless cautioned that these
findings still require experimental confirmation before a bi-phasic effect of lead on peri-
pheral nervous functions can be assumed.
12.4.3 Animal Studies
The following sections focus on recent experimental studies of lead effects on behav-
ioral, morphological, physiological, and biochemical parameters of nervous system development
and function in laboratory animals. Several basic areas or issues are addressed: (1) behav-
iorial toxicity, including the question of critical exposure periods for concurrent induction
or later expression of behavioral dysfunction in motor development, learning performance, and
social interactions; (2) alterations in morphology, including synaptogenesis, dendritic deve-
lopment, myelination, and fiber tract formation; (3) perturbations in various electrophysiolo-
gical parameters, e.g., ionic mechanisms of neurotransmission or nerve conduction velocities
in various tracts; (4) disruptions of biochemical processes such as energy metabolism and
chemical neurotransmission; (5) the persistence or reversibility of the above types of effects
beyond the cessation of external lead exposure; and (6) the issue of "threshold" for neuro-
toxic effects of lead.
Since the initial description of lead encephalopathy in the developing rat (Pentschew and
Garro, 1966), considerable effort has been made to define more closely the extent of nervous
system involvement at subencephalopathic levels of lead exposure. This experimental effort
has focused primarily on exposure of the developing organism. The interpretation of a large
number of studies dealing with early iji vivo exposure to lead has, however, been made diffi-
cult by variations in certain important experimental design factors across available studies.
One of the more notable of the experimental shortcomings of some studies has been the
occurrence of undernutrition in experimental animals (U.S. Environmental Protection Agency,
12-110
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1977). Conversely, certain other studies of lead neurotoxicity in experimental animals have
been confounded by the use of nutritionally fortified diets, i.e., most commercial rodent
feeds (Michaelson, 1980). In general, deficiencies of certain minerals result in increased
absorption of lead, whereas excesses of these minerals result in decreased uptake (see Chapter
10). Dietary mineral and vitamin components are known to alter certain neurotoxic effects of
lead (Woolley and Woolley-Efigenio, 1983). Commercial feeds may also be contaminated by vari-
able amounts of heavy metals, including as much as 1.7 ppm of lead (Michaelson, 1980). Ques-
tions have also been raised about possible nutritional confounding due to the acetate radical
in the lead acetate solutions often used as the source of lead exposure in experimental animal
studies (Barrett and Livesey, 1982).
Another important factor that varies among many studies is the route of exposure to lead.
Exposure of the suckling animal via the dam would appear to be the most "natural" method, yet
may be confounded by lead-induced chemical changes in milk composition. On the other hand,
intragastric gavage allows one to determine precisely the dose and chemical form of admin-
istered lead, but the procedure is quite stressful to the animal and does not necessarily re-
flect the actual amount of lead absorbed by the gut. Injections of lead salts (usually per-
formed intraperitoneally) do not mimic natural exposure routes and can be complicated by local
tissue calcinosis at the site of repeated injections.
Another variable in experimental animal studies that merits attention concerns species
and strains of experimental subjects used. Reports by Mykkanen et al. (1980) and Overmann et
al. (1981) have suggested that hooded rats and albino rats may differ in their sensitivity to
the toxic effects of lead, possibly because of differences in their rates of maturation and/or
rates of lead absorption. Such differences may account for variability of lead's effects and
differences in exposure-response relationships between different species as well.
Each of the above factors may lead to widely variable internal lead burdens in the same
or different species exposed to roughly comparable amounts of lead, making comparison and in-
terpretation of results across studies difficult (Shellenberger, 1984). The force of this
discussion, then, is to emphasize the importance of measurements of blood and tissue concen-
trations of lead in experimental studies. Without such measures, attempts to formulate dose-
response relationships are futile. This problem is particularly evident in later sections
dealing with the morphological, biochemical, and electrophysiological aspects of lead neuro-
toxicity. Jji vitro studies reviewed in those sections, in contrast to iji vivo studies, are of
limited relevance in dose-response terms. The in vitro studies, however, provide valuable
information on basic mechanisms underlying the neurotoxic effects of lead.
The following sections discuss and evaluate the most recent studies of nervous system in-
volvement at subencephalopathic exposures to lead. Most of the older studies are reviewed in
12-111
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the previous Air Quality Criteria Document for Lead (U.S. Environmental Protection Agency,
1977).
12.4.3.1 The Behavioral Toxicity of Lead: Critical Periods for Induction and Expression of
Effects. The perinatal period of ontogeny has been generally recognized as a particularly
critical time for the initiation of neurobehavioral pertubations by exposure to lead (U.S.
Environmental Protection Agency, 1977; Reiter, 1982; Kimmel, 1984). This view is based in
part on the metabolic characteristics of young organisms, which show comparatively greater
absorption and retention of lead (see Chapter 10). In addition, a number of behavioral
studies have compared the effects of lead exposure at different times during ontogeny and have
often found effects associated only with perinatal exposure (e.g., Brown, 1975; Brown et al.,
1971; Padich and Zenick, 1977; Shigeta et al., 1977; Snowdon, 1973).
On the other hand, several studies have demonstrated that alterations in behavior can
result from exposure after weaning or maturation in rats (Angell and Weiss, 1982; Bushnell and
Levin, 1983; Cory-Slechta and Thompson, 1979; Geist and Mattes, 1979; Geist et al., 1985;
Kowalski et al., 1982; Lanthorn and Isaacson, 1978; McLean et al., 1982; Nation et al., 1982;
Ogilvie and Martin, 1982; Shapiro et al., 1973). Similar findings have been noted in adult
subjects of other species, including pigeons (Barthalmus et al., 1977; Dietz et al., 1979) and
fish (Weir and Mine, 1970).
The fact that late developmental exposure to lead can induce behavioral effects in
animals does not mean, of course, that early exposure is less effective or important. As the
following sections will show, the toxic effects of lead may be induced at various stages of
life, with the expression of these effects following closely or, in some cases, after con-
siderable delay.
12.4.3.1.1 Development of motor function and reflexes. A variety of methods have been used
to assess the effect of lead on the ability of experimental animals to respond appropriately
either by well-defined motor responses or gross movements, to a defined stimulus. Such res-
ponses have been variously described as reflexes, kineses, taxes, and "species-specific" be-
havior patterns. The air righting reflex, which refers to the ability to orient properly with
respect to gravity while falling through the air and to land on one's feet, is only one of
several commonly used developmental markers of neurobehavioral function (Tilson and Harry,
1982). Overmann et al. (1979) found that development of this particular reflex was slowed in
rat pups exposed to lead via their dams (0.02 or 0.2 percent lead acetate* in the dams' drink-
ing water). However, neither the auditory startle reflex nor the ability to hang suspended by
the front paws was affected.
""Concentrations are presented here as originally reported by authors. Note that a 0.2 percent
solution of lead acetate contains 0.1 percent lead. Also, for comparative purposes, a con-
centration of 0.1 percent equals 1000 ppm.
12-112
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Grant et al. (1980) exposed rats indirectly to lead iji utero and during lactation through
the mothers' milk and, after weaning, directly through drinking water containing the same lead
concentrations their respective dams had been given. In addition to morphological and physi-
cal effects [see Sections 12.5, 12.6, and 12.8 for discussions of this work as reported by
Fowler et al. (1980), Kimmel et al. (1980), Faith et al. (1979), and Luster et al. (1978)],
there were delays in the development of surface righting and air righting reflexes in subjects
exposed under the 50- and 250-ppm lead conditions; other reflexive patterns showed no effect.
Locomotor development generally showed no significant alteration due to lead exposure, but
body weight was significantly depressed for the most part in the 50- and 250-ppm pups.
Rabe et al. (1985) used a similar experimental paradigm to evaluate developmental land-
marks in rat pups exposed via their dams to a 0.5 percent lead acetate solution. Although
this concentration of lead was much higher than the drinking water solutions used by Grant
et al. (1980), Rabe et al. (1985) found no apparent delays in the development of surface
righting and negative geotaxis reflexes, nor in the age at which the pups' eyes opened. Body
weight of the pups was reduced slightly at birth, but by postnatal day (PND) 30 the lead-
exposed pups had attained normal average weight. In comparing these two studies, it should
also be noted that the mean blood lead level at PND 16 was only 20 ug/dl for pups exposed
indirectly to 2500 ppm lead by Rabe et al. (1985), as opposed to a median of 35 ug/dl at PND
11 for pups exposed indirectly to 50 ppm lead by Grant et al. (1980). These differences are
probably attributable to the different diets used in the studies (see Mylroie et al., 1978).
The ontogeny of motor function in lead-exposed rat pups was also investigated by Overmann
et al. (1981). Exposure was limited to the period from parturition to weaning and occurred
through adulteration of the dams' drinking water with lead (0.02 or 0.2 percent lead acetate).
The development of swimming performance was assessed on alternate days from PND 6 to 24. No
alterations in swimming ability were found. Rotorod performance was also tested at PND 21,
30, 60, 90, 150, and 440. Overall, the ability to remain on a rotating rod was significantly
impaired (p <0.01) at 0.2 percent and tended to be impaired (0.10 > p > 0.05) at 0.02 percent
(blood lead values were not reported). However, data for individual days were statistically
significant only on PND-60 and 150. An adverse effect of lead exposure on rotorod performance
at PND 30-70 was also found in an earlier study by Overmann (1977) at a higher exposure level
of 30 mg/kg lead acetate by intubation (average blood lead value at PND 21: 173.5 ± 32.0
ug/dl). At blood lead concentrations averaging 33.2 ± 1.4 ug/dl, however, performance was not
impaired. Moreover, other studies with average blood lead concentrations of approximately 61
ug/dl (Zenick et al., 1979) and 30-48 ug/dl (Grant et al., 1980) have not found significant
effects of lead on rotorod performance when tested at PND 21 and 45, respectively. Compari-
sons between studies are confounded by differences in body weight and age at time of testing
and by differences in speed and size of the rotorod apparatus (Zenick et al., 1979).
12-113
-------
Kishi et al. (1983) evaluated reflex development and motor coordination in male rats ex-
posed to lead acetate by gavage on PND 3-21. The air righting reflex was significantly
delayed in all three lead exposure groups (the lowest level producing an average blood lead
level of 59 ug/dl at PND 22). The startle reflex showed no effect, and eye opening was accel-
erated in the lowest exposure group. Rotorod performance at PND 53-58 was significantly
impaired in the highest exposure group (average blood lead level: 186 ug/dl at PND 22).
Ambulation was assessed at PND 59-60 and showed a high degree of variability across the lead
exposure groups (very low or very high levels or movement); other measures of activity showed
no differences. The effects on ambulation were not evident at PND 288-289.
Delays in the development of gross activity in rat pups have been reported by Crofton et
al. (1980) and by Jason and Kellogg (1981). It should be noted that very few studies have
been designed to measure the rate of development of activity. Ideally, subjects should be
assessed daily over the entire period of development in order to detect any changes in the
rate at which a behavior pattern occurs and matures. In the study by Crofton et al. (1980),
photocell interruptions by pups as they moved through small passageways into an "exploratory
cage" adjacent to the home cage were automatically counted on PND 5-21. Pups exposed i_n utero
through the dams' drinking water (200 mg/1 solution of lead chloride) lagged controls by ap-
proximately one day in regard to characteristic changes in daily activity count levels start-
ing at PND 16. (Blood lead concentrations at PND 21 averaged 14.5 ± 6.8 ug/dl for represen-
tative pups exposed to lead j_n utero and 4.8 ± 1.5 ug/dl for controls.) Another form of
developmental lag in gross activity around PND 15-18, as measured in an automated activity
chamber, was reported by Jason and Kellogg (1981). Rats were intubated on PND 2-14 with lead
at 25 mg/kg (blood lead: 50.07 ± 5.33 ug/dl) and 75 mg/kg (blood lead: 98.64 ± 9.89 ug/dl).
In this case, the observed developmental lag was in the characteristic decrease in activity
that normally occurs in pups at that age (Campbell et al., 1969; Melberg et al., 1976); thus,
lead-exposed pups were significantly more active than control subjects at PND 18.
One question that arises when ontogenetic effects are discovered concerns the possible
contribution of the lead-exposed dam to her offsprings' slowed development through, for exam-
ple, reduced or impaired maternal caregiving behavior. A detailed assessment of various
aspects of maternal behavior in chronically lead-exposed rat dams by Zenick et al. (1979)f
discussed more fully in Section 12.4.3.1.4, and other studies using cross-fostering techniques
(Crofton et al., 1980; MykkMnen et al., 1980) suggest that the deleterious effects observed in
young rats exposed to lead via their mothers' milk are not ascribable to alterations in the
dams' behavior toward their offspring. Chronically lead-exposed dams may, if anything, tend
to respond adaptively to their developmentally retarded pups by, for example, more quickly
retrieving them to the nest (Davis, 1982) or nursing them for longer periods (Barrett and
Livesey, 1983).
12-114
-------
12.4.3.1.2 Locomotor activity. The spontaneous activity of laboratory animals has been meas-
ured frequently and in various ways as a behavioral assay in pharmacology and toxicology
(Reiter and MacPhail, 1982). Such activity is sometimes described as gross motor activity or
exploratory behavior, and is distinguished from the motor function tests noted in the previous
section by the lack of a defined eliciting stimulus for the activity. With reports of hyper-
activity in lead-exposed children (see Section 12.4.2), there has naturally been considerable
interest in the spontaneous activity of laboratory animals as a model for human neurotoxic ef-
fects of lead (see Table 12-3). As a previous review (U.S. Environmental Protection Agency,
1977) of this material demonstrated, however, and as other reviews (e.g., Jason and Kellogg,
1980; Michaelson, 1980; Mullem'x, 1980) have since confirmed, the use of activity measures as
an index of the neurotoxic effects of lead has been fraught with difficulties.
First, there is no unitary behavioral endpoint that can be labeled "activity." Activity
is, quite obviously, a composite of many different motor actions and can comprise diverse be-
havior patterns including (in rodents) ambulation, rearing, sniffing, grooming, and, depending
on one's operational definition, almost anything an animal does. These various behavior pat-
terns may vary independently, so that any gross measure of activity which fails to differen-
tiate these components will be susceptible to confounding. Thus, different investigators'
definitions of activity are critical to interpreting and comparing these findings. When these
definitions are sufficiently explicit operationally (e.g., activity as measured by rotations
of an "activity wheel"), they are frequently not comparable with other operational definitions
of activity (e.g., movement in an open field as detected by photocell interruptions). More-
over, empirical comparisons (e.g., Capobianco and Hamilton, 1976; Tapp, 1969) show that dif-
ferent measures of activity do not necessarily correlate with one another quantitatively.
In addition to these rather basic difficulties, activity levels are influenced greatly by
numerous variables such as age, sex, estrous cycle, time of day, novelty of environment, and
food deprivation. If not controlled properly, any of these variables can confound measure-
ments of activity levels. Also, nutritional status has been a frequent confounding variable
in experiments examining the neurotoxic effects of lead on activity (see reviews by U.S.
Environmental Protection Agency, 1977; Jason and Kellogg, 1980; Michaelson, 1980). In
general, it appears that rodents exposed neonatally to sufficient concentrations of lead ex-
perience undernutrition and subsequent retardation in growth; as Loch et al. (1978) have
shown, retarded growth per s_e can induce increased activity of the same type that has been
attributed to lead alone in some earlier studies.
In view of the various problems associated with the use of activity measures as a behav-
ioral assay of the neurotoxic effects of lead, the discrepant findings summarized in Table
12-115
-------
TABLE 12-3. EFFECTS OF LEAD ON ACTIVITY IN RATS AND MICE
Increased
Decreased
Age-dependent,
qualitative, mixed or
no change
Baraldi et al. (1985)
Czech and Hoi urn (1984)
Driscoll and Stegner (1978)
Goiter and Michael son (1975)
Kostas et al. (1976)
Overmann (1977)
Petit and Alfano (1979)
Sauerhoff and Michael son
(1973)
Silbergeld and Goldberg
(1973, 1974a,b)
Weinreich et al. (1977)
Winneke et al. (1977)
Booze et al. (1983)
Driscoll and Stegner (1976)
Flynn et al. (1979)
Gray and Reiter (1977)
Reiter et al. (1975)
Verlangieri (1979)
Alfano and Petit (1985)
Barrett and Livesey (1982, 1985)
Brown (1975)
Collins et al. (1984)
Crofton et al. (1980)
Dolinsky et al. (1981)
Dubas and Hrdina (1978)
Geist and Balko (1980)
Geist and Praed (1982)
Grant et al. (1980)
Gross-Selbeck and
Gross-Selbeck (1981)
Hastings et al. (1977)
Jason and Kellogg (1981)
Kishi et al. (1983)
Kostas et al. (1978)
Krehbiel et al. (1976)
Loch et al. (1978)
McCarren and Eccles (1983)
Minsker et al. (1982)
Mullenix (1980)
Ogilvie and Martin (1982)
Rabe et al. (1985)
Rafales et al. (1979)
Schlipkb'ter and Winneke
(1980)
Shimojo et al. (1983)
Sobotka and Cook (1974)
Sobotka et al. (1975)
Zimering et al. (1982)
12-116
-------
12-3 should come as no surprise. Until the measurement of "activity" can be better standar-
dized, there appears to be little basis for comparing, or utility in further discussing, the
results of studies listed in Table 12-3.
12.4.3.1.3 Learning ability. When animal learning studies related to the neurotoxic effects
of lead were reviewed in 1977 (U.S. Environmental Protection Agency, 1977), a number of criti-
cisms of existing studies were noted. A major limitation of early work in this field was the
lack of adequate information on the exposure regimen (dosage of lead, how precisely adminis-
tered, timing of exposure) and the resulting body burdens of lead in experimental subjects
(concentrations of lead in blood, brain, or other tissue; time course of blood or tissue lead
levels; etc.). A review of studies appearing since 1977 reveals a notable improvement in this
regard. A number of more recent studies have also attempted to control for the confounding
factors of litter effects and undernutrition--variables that were generally not controlled in
earlier studies.
Unfortunately, other criticisms are still valid today. The reliability and validity of
behavioral assays remain to be established adequately, although progress is being made. The
reliability of a number of common behavioral assays for neurotoxicity is currently being de-
termined by several independent U.S. laboratories (Buelke-Sam et al., 1985). The results of
this program should help standardize some behavioral testing procedures and perhaps create
some reference methods in behavioral toxicology. Also, as well-described studies are repli-
cated within and between laboratories, the reliability of certain experimental paradigms for
demonstrating neurotoxic effects is effectively established.
Some progress is also being made in dealing with the issue of the validity of animal be-
havioral assays. As the neurological and biochemical mechanisms underlying reliable behav-
ioral effects become better understood, the basis for extrapolating from one species to
another becomes stronger and more meaningful. An awareness of different species' phylogene-
tic, evolutionary, and ecological relationships can also help elucidate the basis for compar-
ing behavioral effects in one species with those observed in another (Davis, 1982).
Tables 12-4 and 12-5 summarize exposure conditions, testing conditions, and results of a
number of recent studies of animal learning (see U.S. Environmental Protection Agency, 1977,
for a summary of earlier studies). The variety of exposure measures and testing paradigms
makes it impossible to organize these studies in a coherent dose-response fashion. Conse-
quently, the tables present, respectively, rodent and primate studies arranged alphabetically
by author and/or chronologically where appropriate. One point of obvious interest is the
lowest level of exposure at which behavioral effects are clearly evident. Such a determina-
tion is best done on a species-by-species basis. Rats seem to be the experimental animal
12-117
-------
TABLE 12-4. RECENT ANIMAL TOXICOLOGY STUDIES OF LEAD'S EFFECTS ON LEARNING IN RODENTS'
a
Reference
Alfano
and Petit
(1985)
Angell
& Weiss
(1982)
rv
i_i
*— »
03
Booze et
al.
(1983)
Bushnell
and Levin
(1983)
Experimental
animal
(species
or strain)
Rat
(L-E)
Rat
(L-E)
Rat
(F-344)
Rat
(S-D)
Lead exposure
cone.
(medium)
0.4 or
4X PbC03
(food)
0.2%
Pb(Ac)2
(water)
3 or 6
ing/ kg
TEL
(15X
ethanol)
10 or
100 ppm
Pb
(water)
period
(route)
PND 1-25
(via dam
and
direct)
PND 3-21
(dam's
milk)
and/or
21-130
(direct)
PND 5,
once
(s.c.)
PND 21-
56
(direct)
Treatment
groups
(n)
C5b (40)
C,0 (50)
Pb, (50)
Pb2 (50)
0-0 (20) ,
0-Pb (20) '
Pb-0 (24) ,
Pb-Pb (24)'
C0C (24)
C15 (23)
Pb, (24)
Pbz (23)
C (6)
Pb, (6)
Pb2 (6)
Litters
per
group
8
5
5
10
5cnl 1 t
I Sp I 1 t
6enl i *•
, Sp M t
random
selection
from 12
litters
?
Tissue lead
(age measured)
See Petit &
Alfano (1979)
for representa-
PbB levels
PbB (130d):
0-0: 2 ug/dl
0-Pb: 66
Pb-0: 9
Pb-Pb: 64
7
Brain- Pbd
(57 d):
C: 0 ug/g
Pb,: 0.05
Pb2: 0.70
Age at
testing
66-
100 d
101-
123 d
58-
130 d
18 d
4, 5,
6, and
7 wk
Testing
paradigm
(task)
Passive
avoidance
(remain in
1 of 2 com-
partments
to avoid
shock);
T-maze
(spontaneous
alternation)
Operant
(multiple
FI-TO-
FR-TO)
Passive
avoidance
(renain
in 1 of 2
compartments
to avoid
shock)
Radial arm
naze (spon-
taneous
alternation)
Non-
behavioral
effects
B.w. of
Cs-Ss >
C0- and
Pb2-Ss
Pb-Pb Ss
sig. lower
b.w. post-
weaning
B.w. of
Pb-Ss
< C-Ss
B.w. of
Pb2-Ss <
C-Ss~
Behavioral
results
Latencies of Pb-Ss
sig. shorter than
C-Ss1; Pb2 latencies
sig. shorter than
C10's. Both Pb
groups performed
sig. less sponta-
neous alternation
than C-Ss.
Groups exposed post-
weaning (0-Pb, Pb-
Pb) had longer
Inter-Response
Tines; group ex-
posed preweaning
(Pb-0) had
shorter IRTs.
Pb,- females showed
sig. poorer reten-
tion of avoidance
than ethanol con-
trols.
Both Pb,- and Pb2-
Ss chose unexplored
amis sig. less often
than C-Ss.
-------
TABLE 12-4. (continued)
Experimental
aninal
(species
Reference or strain)
Cory- Rat
Slechta (S-D)
&
Thompson
(1979)
Cory- Rat
Slechta (S-D)
et al.
(1981)
i — '
no
i
t— •
Cory- Rat
Slechta (L-E)
et al.
(1983)
Lead exposure
cone.
(Dediua)
1) 50,
2) 300,
or
3) 1000
pp*
Pb(Ac)2
(water)
100 or
300 ppn
Pb(Ac)2
(water)
50, 100
or 500
Pb(Ac)2
(water)
period
(route)
PNO 20-
a) 70 or
b) 150
(direct)
PND
21-?
PND 21-
a) 158 or
b) 315
(direct)
Treatment
groups
(n)
la: .
C (4)e
Pb (5)
Ib:
C (4)e
Pb (6)
2b:
C (3)e
Pb (4)
3b: „
C (4)e
Pb (5)
C (4)
Pbi (5)
Pb2 (5)
C (6)
Pbt (6)
Pb2 (6)
Pb3 (6)
Litters
per
group
random
assign-
Kent
randoH
assign-
ment
randoa
assign-
Kent,
balanced
for b.w.
Tissue lead
(age measured)
PbB (150 d):
C: ~6 jjg/dl
la: -3
Ib: -7
2b: -27
3b: -42
Brain-Pb
(post-test):
C: 14-26 ng/g
Ph,: 40-142
Pb2: 320-1080
PbB (max):
C: <2 ug/dl
Pbi: -40
Pb2: -50
Pb3: -90
Brain-Pb
(336 d):
C: 0.01 (jg/g
Pb,: 0.31
Pb2: 0.57
Pb3: 1.4
Testing Non-
Age at paradigm behavioral
testing (task) effects
55- Operant None
140 d (Fl-30 sec)
55- Operant None
? d (mini BUB
duration
bar-press)
55 d Operant None
(FI-1 m1n)
Behavioral
results
Increased response
rate and inter- S
variability in both
Pbi . and Pb2
groups; decreased
response rate in Pb3
group; effects in
Pb! reversed after
exposure terminated.
Pb groups impaired:
decreased response
durations; increased
response latencies;
failure to improve
performance by
external stimulus
control .
Higher response
rates in Pb-Ss;
number of sessions
to reach max.. rate
a direct function
of Pb expos. Early
vs. late teraina-
nation of exposure
period produced no
difference in re-
sponse rates.
sponse rates.
-------
TABLE 12-4. (continued)
Experimental
aniaal
(species
Reference or strain)
Cory- Rat
Slechta (L-E)
et al.
(1985)
Dietz Rat
et al. Expt. l(L-E)
(1978)
Expt. 2(CD)
i— *
no
i
i— •
I\5
O
Flynn Rat
et al. (L-E)
(1979) Expt. 1
Expt. 2
Expt. 3
Lead exposure Treatment
cone.
(BediUM)
25 pp.
Pb (Ac)2
(water)
200
•gAg
Pb(Ac)2
(gavage)
250 ppa
Pb
(water)
0.5X
Pb(Ac)2
(water)
0.2X
Pb(Ac)2
(water).
225 •gAg
Pb
(gavage),
0.25X
Pb(Ac)2
(water)
saae
as above
except 90
•gAg Pb
(gavage)
period
(route)
PND 21-
tenaina-
nation
(direct)
PND
3-30
(direct)
Preconcep-
tion to
termination
(via da*
until weaning
then direct)
Preconception
- PND 22
(via daa)
Preconception
- birth
(via daa).
birth -
weaning
(direct).
weani ng
- termination
(direct)
saae as
above except
stopped at
PHD 33
groups
(n)
C (12)
Pb (12)
C (6) ,
Pb (7)j
C (4)e
Pb (4)
1
C (8)
Pb (10)
C (12)
Pb (12)
C (10) ,
Pb (10)J
Litters
per Tissue lead Age at
group (age Measured) testing
randon PbB (99, 143, 50 d
assign- 186 d):
Bent, C: <1 ug/dl
balanced Pb: 15-20
for weight
2 snlit ? 3 "°
2. split or
21 mo
? ? 8 no
8 Brain-Pb ?
10 (3 d):
C: -0
Pb: 0. 174 ug/g
(30-34 d):
no sig. diffs.
6 (75-76 d): 49-
6 C: 0.13 ug/g 58 d
Pb: 1.85
. see above 58-
* 60 d
Testing Non-
paradig* behavioral
(task) effects
Operant
(FI-1 win)
Operant
(aininuB
20-sec
between
bar-presses)
Radial
am aaze
(spontaneous
alternation)
Passive
avoidance
(reaain
in 1 of 2
coBpartaents
to avoid
shock)
Shuttle-box
signalled
avoidance
(BOVC fro* one
co*part»ent to
other to avoid
elect, shock)
None
None
Pb-Ss b.w.
lower 1 wk.
prior to
test; C-Ss
reduced to
same wt.
at test.
Brain wts.
of Pb-Ss
< C-Ss;
no other
differences.
None
Non*
Behavioral
results
Sig. higher response
rate and shorter
IRTs by Pb-Ss during
first 40 sessions.
Short IRTs (S4 sec)
more prevalent in
Pb-Ss than in
C-Ss", but did not
result in different
reward rates; Pb-Ss
showed higher varia-
bility in response
rate under d-aaphet-
aBine treataent.
No sig. difference
between Pb-Ss
and C-Ss.
No sig. difference
in trials to crite-
rion, but Pb-Ss Bade
sig. fewer partial
excursions froa
"safe" coapartaent.
No sig. difference
between Pb-Ss
and C-Ss.
-------
TABLE 12-4. (continued)
Reference
Geist
& Mattes
(1979)
Geist
et al.
(1985)
t— >
ro
i
i — >
ro
i— •
Gross-
Selbeck
& Gross-
Selbeck
(1981)
Hastings
et al.
(1977)
Experimental
ani*al
(species
or strain)
Rat
(S-0)
Rat
(S-D)
Rat F,
00
F2
Rat
(L-E)
Lead exposure Treatment
cone.
(medium)
25 or
50 pp.
Pb(Ac)2
(water)
25 or
SO pp.
Pb(Ac)2
(water)
i a/kg
Pb(Ac)2
(food)
II
109 or
545 ppm
Pb(Ac)2
(water)
period
(route)
MM) 23-
termi nation
(direct)
PND 21-
65
(direct)
Postweaning
- termination
(direct)
Preconception
- weaning
(via dam)
PNO 0-
21
(dan's
•ilk)
groups
(n)
C (7)
Pbt (7)
Pb2 (7)
C (6)
Pbj (6)
Pb2 (6)
C (6)
Pb (6)
C (6)
Pb (6)
C (12)
Pbj (12)
Pb2 (12)
Litters
per Tissue lead
group (age measured)
? ?
? ?
? PbB
(-180 d):
C: 6.2 ug/dl
Pb: 22.7
? (-110 d):
C: 3.7
Pb: 4.6
randon PbB
selection (20 d):
fron 9 C: 11 ug/dl
litters Pbj: 29
Pb2: 42
(60 d):
C: 4
Pbt: 5
Pba: 9
Testing Non-
Age at paradign behavioral
testing (task) effects
58-? d Hebb- None
Williams
maze
(find way
to goal
box)
61 d T-«aze None
( spontaneous
alternation);
143 d Hebb- Will Jains
naze (find
way to goal
box)
Adult Operant None
(DRH)
3-4 BO
-90- Operant None
186 d (successive
brightness
discrin. )
Behavioral
results
Pbi- and Pb2-Ss
•ade sig. More
errors than C-Ss;
Pb2-Ss slower
thaiTC-Ss to
traverse maze.
Rate of spontaneous
alternation sig.
reduced in Pb-Ss.
No sig. differences
in H-W naze except
except for shorter
latency of Pb-Ss to
leave start box.
Both F! and F2
(especially F2)
Pb-Ss had greater
X rewarded responses
than C-Ss, i.e. ,
Pb-Ss bar-pressed
at higher rate
than C-Ss.
No sig. differences
between Pb-Ss
and C-5s in"
learning original
or reversed
di scrim, task.
-------
TABLE 12-4. (continued)
Reference
Hastings
et al.
(1979)
Hastings
et al.
(1984)
i— •
PO
i — »
rO
ro
Kishi
et al.
(1983)
Kowalski
et al.
(1982)
Experimental
animal
(species
or strain)
Rat
(L-E)
Rat
(L-E)
Rat
(W)
House
(W)
Lead exposure
cone.
(medium)
0.02
or
0.2X
Pb(Ac)2
(water)
0.10X
or 0.20X
Pb(Ac)2
(water)
45, 90,
or 180
M9/9
b.w.
(water)
2 pp. Pb
(water)
period
(route)
?ND 0-21
(dan's
•ilk)
PHD 0 to
a) 30
(dai's
•ilk)
or
b) term-
nation
(direct)
PHD 3-21
(gavage)
Adult
(direct)
Treatment
groups
(n)
C (23)
Pbt (11)
Pb2 (13)
C (22)
Pbt (25)
Pb2_ (22)
Pb2? (23)
D
C (10)
Pbi (10)
Pt>2 (10)
Pb3 (9)
C (16)
Pb (16)
Litters
per
group
random
selection
from 15
litters
random
selection
fro* 49
litters
random
selection
1
Tissue lead
(age Measured)
PbB (20 d):
C: 11 ug/dl
Pbt: 29
Pb2: 65
Brain- Pb
(20 d):
C: 12.5 ug%
Pb1= 29
Pb2: 65
PbB
(20 d):
C: 3 ug/dl
Pbt: 30
Pb2 : 57
Pb2': 40
(90Dd):
C: 5
Pb^ 31
Pb2 : 9
Pb2a: 42
PbB (22d):
C: 10 ug/dl
Pbt: 59
Pb2: 152
Pb3: 186
?
Age at
testing
120 d
270 d
330 d
-90 d
75-270 d
Adult
(13 d
after
start of
exposure)
Testing Hon-
paradign behavioral
(task) effects
1) Operant None
(simult. vis.
di scrim. )
2) T-maze
(success, vis.
discria.)
3) Operant
(go/no-go task)
Operant None
(1) spatial
discr. with
successive
reversal s ;
2) siault.
visual discr.)
Operant B.w. of Pb3-
(1) CRF Ss < C-Ss
2) FR 20
3) EXT
4) DRL 20- sec
5) EXT)
Water T-*aze None
(spatial
discria. )
Behavioral
results
Pb2-Ss sig. slower
to reach criterion
than C-Ss on
simultaneous visual
discrimination task;
no sig. differences
on successive and
go/no-go discria.
tasks.
No sig. differences
in performance except
for sig. pos. corre-
lation between day-20
PbB levels and number
of non- rewarded re-
sponses between trials.
Sig. greater varia-
bility in CRF re-
sponding by Pt>! and
Pb3-Ss. No sig.
differences in
mean response
rates except Pbt-
Ss better than C-
5s at end of DRL
training.
Pb-Ss made more
errors than C-Ss;
food deprivation
exacerbated effect.
-------
TABLE 12-4. (continued)
Reference
Lanthorn &
Isaacson
(1978)
He Lean
et al.
(1982)
£Hilar
i et al.
£ (1981i>)
CO
Nation
et al.
(1982)
Experiwntal
aninal
(species
or strain)
Rat
(L-E)
House
(Swiss)
Rat
(L-E)
Rat
(S-D)
Lead exposure
cone.
(MediuM)
0.27X Pb
(water)
20 or
2000 ppM
Pb
(water)
25. 100,
or 200
•g/kg b.w.
Pb
(gavage)
10 Mg/kg
b.w. Pb
(food)
period
(route)
Adult
(direct)
Adult
(direct)
PMO 4-31
(direct)
PMD 100-
terai na-
tion
(direct)
Treatment
groups
(n)
C (4)
Pb (6)
C (16)
Pb, (16)
Pbj (16)
C (10)
Pb» (5)
Pb2 (4)
Pb3 (6)
C (8)
Pb (8)
Litters
per Tissue lead
group (age Measured)
7 7
? ?
3 PbB (32 d):
4 C: 5 ug/dl
4 Pb,: 26
4 Pb2: 63
Pb3: 123
7 7
Age at
testing
Adult
Adult
(10 d
after
start of
exposure)
50 d
156 d
Testing
paradign
(task)
T-Maze
(1) spontane-
ous alterna-
tion
2) light
discriM.
3) spatial
discriM.)
Water T-naze
(spatial
discriM.)
Operant
(spatial
alternation
levers)
Operant
(conditioned
suppression
of respond-
Non-
behavioral
effects
C-Ss
pan— fed
to control
for loss
of b.w.
None
Sig. slower
growth rate
in Pb3-Ss
None
ing on multiple
OverMann
(1977)
Rat
(L-E)
10, 30,
or 90
Pb(Ac)2
(gavage)
PHO 3-21
(direct)
C
Pbt 12-
Pb2 25
Pb3 ea.
? PbB (21 d):
C: 15 ug/dl
Pbt: 33.2
Pb2: 173.5
Pb3: 226.1
26-29 d
67-89 d
79-101 d
83-105 d
95-117 d
VI schedule)
Avers ive
conditioning
(1) active
2) passive)
Operant
(inhibit
response)
E-maze
di scrim. :
(1) spatial
2) tactile
3) visual)
None
Behavioral
results
Pb-Ss had sig.
lower rate of
spontaneous alterna-
tion; Pb-Ss sig.
slower than C-Ss
only on 1st spatial
discrin. task.
Pb-Ss showed no
i«prove»ent in
performance coir-
pared to C-Ss.
No sig. differences
between C-Ss
and Pb-Ss.
Presentation of tone
associated with
electrical shock
disrupted steady-
state responding
More in PB-Ss than
in C-Ss.
Pb3-Ss sig. slower
in acquisition and
extinction of active
avoidance response;
no sig. diffs. for
passive avoidance.
All Pb groups failed
to inhibit responses
Pb2 3-Ss sig. worse
than C-Ss only on
tactile discrim.
-------
TABLE 12- C-Ss;
gross
toxicity
in Pb2-Ss;
lower brain
wts. in
Pbt-Ss
B.w. of
Pb-Ss <
C-Ss at
birth
(includes
pair- fed
C2-Ss)
Behavioral
results
Pb-Pb group
had sig. fewer
rewarded responses
and took sig. longer
to complete FR 20
requirement.
Pb-Ss showed
slower acquisition
(in terms of speed
and rate, but not
errors); by 3rd
session, no sig.
diffs. except that
Pb-Ss made sig.
fewer errors than
C-Ss.
No sig. diff.
between Pb- and O
Ss in naze learning;
Tsol at i on- reared
Pb-Ss less success-
ful than C.-Ss
on passive-avoidance
task; enrichment-
reared Pbj-Ss = C-Ss
but Pb2 -Ss sig.
worse on passive
avoidance.
No sig. diffs. in
acquisition or
reversal errors.
-------
TABLE 12-4. (continued)
Experimental
animal
(species
Reference or strain)
Rosen Rat
et al. (L-E)
(1985)
,_,
PO
t
! — '
in
Schlipkbter Rat
& Winneke (?)
(1980) Expt. 1
Expt. 2
Cwn+ ^
txpt . 3
Expt. 4
Lead exposure Treatment Litters
cone.
(nediun)
10 ng/kg
b.w.
Pb(Ac)2
25pp.
Pb
(food)
75 ppm
Pb
(food)
Sane as
25 or
75 ppm
Pb
(food)
period groups per
(route) (n) group
PND 1-20. Ci (16) •> . ,.
daily Pb, (15) ' ' p
(i.p.) C2 (8) , - ,.
Pb2 (8) ' 2> Sp1lt
Preconception C (10) ?
- PND 120 Pbt (18)
(via dan)
and direct)
a) Prenatal- C (10) ?
7 mo (via Pb2 (10)
dam and Pb2b (10)
di rect
b) Prenatal -
weaning
(via dan)
Expt. 2 C (14) ?
Pb3a (14)
Prenatal C (10) ?
- 7 mo Pb« (10)
(via dam Pb4b (10)
and direct)
Tissue lead
(age measured)
PbB (21 d):
C,: 3 M9/dl
Pb,: 158
(180 d):
C2: 5
Pb2: 8
PbB:
all C: <5 ng/dl
Pbj (21 d):
39.5
(4- mo):
12.0
Pb2 :
(21ad) 29.2
(7 mo) 27.0
Pb2.:
(21Dd) 29.2
(7 mo) 5.2
Pb3 :
(21ad) 29.9
(7 mo) 30.8
(21bd) 29.9
(7 mo) 1.8
(120 d)
Pbt -. 17.8
Pb4*: 28.6
Testing Non-
Age at paradigm behavioral
testing (task) effects
1) 30- Radial-arm B.w. of
50 d maze (find Pb-Ss <
and/or food in each C-Ss at
2) 150 d of 8 arms); 25 d. but
passive not sig.
avoidance after 50 d
(remain in
1 of 2 com-
partments to
avoid shock)
7 mo Lashley ?
jumping
stand
(size
di scrim. )
II II f
II II •>
" Water ?
maze
(spatial
di scrim. )
Behavioral
results
No sig. differences
on radial arm naze
for either Pbj
(young) or Pb2
(adult) Ss. Sig.
longer latency on
passive avoidance
for Pbj-Ss, but not
when retested as
adults. Pb2-Ss
tested first time
at 150 d had sig.
shorter latencies
(i.e. , performed
worse than C-Ss).
Sig. increase in
error repetition
by Pb,-Ss.
Non-sig. (p <0.10)
increase in error
repetition by Pb2-
Ss.
No sig. differences
between Pb3-Ss
and C-Ss.
35% of Pb4-Ss failed
to reach criterion
(vs. 10X C-Ss); 35%
also failed retest
after 1 wk (vs. OX
C-Ss).
-------
TABLE 12-4. (continued)
Reference
Taylor
et al.
(1982)
Winneke
et al.
(1977)
t— »
IV>
t— *
IS>
cpl
Winneke
et al.
(1982D)
Zenick
et al.
(1978)
Experiment!
animal
(species
or strain!
Rat
(CD)
Rat
(W)
Rat
(W)
Expt. 1
Expt. 2
Rat
(CD)
il
Lead exposure Treatment
cone.
) (medium)
200 or
400 »g/l
Pb(Ac)2
(water)
1.389
Pb(Ac)2
per kg
diet
(food)
80, 250
or 750
pp* Pb
(food)
-Continuation
1000
•g/kg
Pb(Ac)2
(water)
period groups
(route) (n)
Preconception
- weaning
(via dam)
Preconception
- testing
(via dan
and
direct)
Preconception
- testing
(via dam
and
direct)
of Expt. 1-
Preconception
- weaning
(via dan)
C (12)
Pbj (16)
Cz (4)
Pb2 (4)
C (20)
Pb (20)
C (16)
Pb, (16)
Pb2 (16)
Pbs (16)
C (10)
Pb2 (10)
Pb3 (10)
C (10)
Pb (10)
Litters
per Tissue lead
group (age measured)
69 PbB (21 d):
89 C: 3.7 ug/dl
29 Pbt: 38.2
29 Pb2: 49.9
? PbB
(random (-16 d):
selection C: 1.7 ug/dl
fron Pb: 26.6
110 male (-190 d)
pups) Pb: 28.5
random ?
selection
frwi 5-6
litters
per condi-
tion
(females ?
dropped;
no Pt>! group
for Expt. 2)
5 ?
5
Testing Non-
Age at paradigm behavioral
testing (task) effects
11 d Runway
(traverse
alley to
reach dan
and dry
suckle)
100- Lashley
200 d jumping
stand
(visual
di scrim.
of stimulus:
Dorientation
2)size)
70- Shuttle-box
100 d signalled
avoidance
(move from
one compart-
ment to avoid
elect, shock)
190- Lashley
250 d jumping stand
(size discrim.
30- Water T-maze
40 d (1) black-white
discrim.
55- 2) shape
63 d discrim. )
None
B.w. of
Pb-Ss > C-Ss;
however, size
of Pb-Ss
1 i tters
< C-S
litters.
ALA-D at
SO d:
C: 7.05 U/l
Pb^ 4.26
Pb2: 1.92
Pb3: 1.18
)
B.w. of
Pb-Ss <
C-Ss from
birth to
50 d.
Behavioral
results
No sig. differences
in acquisition of
response, but
both Pb groups
sig. slower to
extinguish when
response no longer
rewarded.
Pb-Ss sig. slower
to learn size
discrimination;
no difference
between Pb and C
groups on orienta-
tion discrim. (a
relatively easy
task).
Expt. 1 Pb-Ss sig.
faster than C-Ss to
learn avoidance
response.
Expt. 2 Pb-Ss
sig. slower than
C-Ss to learn
size discrim.
On both discrim.
tasks, Pb-Ss
made sig. more
errors with sig.
shorter response.
-------
TABLE 12-4. (continued)
Reference
Zenick
et al.
(1979)
Experimental
animal Lead exposure Treatment Litters
(species cone. period groups per
or strain) (medium) (route) (n) group
Rat 750 Preconception 0-0 (?) 5
(CD) ag/kg to Pb-0 (?) 5
Pb(Ac)2 a) weaning Pb-Pb (?) 5
(water) (via dam)
or
b) termination
(via dam
and direct)
Tissue lead
(age measured)
?
Testing
Non-
Age at paradigm behavioral Behavioral
testing (task)
42-? d Operant
(FI-1 min)
effects results
B.w. of Pb-Pb group had sig.
Pb-Ss fewer rewarded
< 0~Ss responses across
from birth sessions than Pb-0
to weaning. or 0-0 groups.
a Abbreviations and symbols:
! — ' -
IX. •
^ ALA-D
rx> b.w.
^ C.
CD
CRF
ORH
DRL
IM
Fj
F-344
FI
FR
i.p.
IRT
r.,-Sc rxntt
information not given in report
delta aninolevulinic acid dehydrase
body weight
control group
substrain of Sprague-Dawley
continuous reinforcement for each response
differential reinforcement of high response rates
differential reinforcement of low response rates
extinction
1st filial generation
2nd filial generation
Fischer-344
fixed interval
fixed ratio
intraperitoneal injection
inter response tine
* fro« litters of 5 mine parh- P.~-^c frnm 1-itforc nf in- hnth
L-E
Pb
Long-Evans
lead-exposed group (subscript indicates exposure level or
other experimental condition)
Pb(Ac)2
PbB
PbC03
PND
S
s.c.
S-D
TEL
TO
U/l
VI
W
WGTA
Ph nrnnnc frnm lit
lead acetate
blood lead
lead carbonate
post-natal day
subject
subcutaneous injection
Sprague Daw ley
tri ethyl lead
time out
unole ALA/min x liter erythrocytes
variable interval
Wistar
Wisconsin general testing
tors nf R parh
apparatus
cC0-Ss sham injected; C15-Ss injected with 15% ethanol.
For Ss on zinc-replete diet; Ss on zinc-deficient diet had higher Pb concentrations.
Height-matched controls.
Pair-fed and -watered controls.
^Inferred from information in report.
-------
TABLE 12-5. RECENT ANIMAL TOXICOLOGY STUDIES OF LEAD'S EFFECTS ON LEARNING IN PRIMATES
Lead exposure
Reference Species
Bushnell Macaca
& Bowman mulatta
(1979a)
Expt. 1
Expt. 2
Test 1
) — '
ro
i
^-j>
ISJ
OO
T_ -* o
I est £.
Test 3
cone.
(medium)
-0.53 or
1.15
mg/kg
Pb C«ilk)
adjusted
to main-
tain tar-
get PbBs
-0.25 or
1.06
rag/kg
Pb (ni Ik)
adjusted
to main-
tain tar-
get PbBs
after
period
(route)
Birth -
1 yr
(direct)
Birth -
1 yr
(direct)
Continual! 01
Treatment
groups
(n)
C (4)
Pbt (3)
Pb2 (3)
C (4)
Pbj (4)
Pb2 (4)
'Continuation of Expt.
n of Expt. 2
exposure terminated at 12 no
Tissue lead
(age measured)
PbB (1st yr)b:
C: ~5 ug/dl
Pbj: 37
Pb2: 58
PbB (1st yr)b:
C: -4 (jg/dl
Pb,: 32
Pb2: 65
PbB (16 mo):
C: ~5 ug/dl
Pb^ 19
Pb2: 46
Testing Non-
Age at paradigm behavioral
testing (task) effects
5- WGTA (form None
10 mo discrim.
reversal
learning)
1.5- 2-choice None
4.5 mo maze
(discr.
reversal
learning)
non- food
reward
5Uf*TA UnnA
WG1 A None
12 mo (series
of 4
reversal
discr.
problems)
15 mo WGTA None
(discr.
reversal
learning,
more
difficult
cues)
Behavioral
results
Both Pb groups
retarded in rever-
sal learning;
Pbz-Ss especially
impaired on 1st
reversal following
overtraining.
Pb2-Ss sig.
retarded on 1st
reversal (confirms
Expt. 1 using dif-
ferent task and
reward to control
for possible con-
founding by motiva-
tional or motor
factors).
Both Pb groups
retarded in
reversal learning;
Pb2-Ss
impaired on 1st
reversals regard-
less of prior over-
training.
Pb2-Ss retarded
on 1st reversal.
-------
TABLE 12-5. (continued)
Reference
Bushnel 1
& Bowman
(1979b)
Mele et
al. (1984)
Levin and
Bowman
Q983)
£ Expt.
ro
§ Expt.
Laughlin
et al.
(1983)
Species
Macaca
mulatta
Macaca
mulatta
Macaca
mulatta
1 (Continuation
2 (Continuation
Macaca
aulatta
Lead exposure
cone. period
(medium) (route)
--Continuation of Bushnel 1
— Continuation of Bushnell
0.29 or Birth -
0. 88 ng/kg 1 yr
Pb (milk) (direct) .
of Expt. 2 of Bushnell &
of Expt. 4 of Bushnell &
3 or 6 Preconception
mg/kg - Birth
Pb(Ac)2 (via mother)
(water)
-10 ng/kg Weeks 5-6
and/or and/or
-0.5 mg/kg birth-
b.w. Pb 1 yr
(•ilk) (direct)
Treatment
groups
(n)
& Bowman (1979a)—
4 Bowman (1979a)—
C (4)
Pb, (3)
Pb2 (3)
C (3)
Pb, (4)
Pb2 (3)
Bowman, 1979a)
Bowman, 1979c)
C (5)
Pbi (3)
Pb2 (4)
C (4)
Phx (4)
Pb2 (4)
Pb3 (4)
Tissue lead
(age measured)
PbB (56 mo):
C: 4 ug/dl
Pbj: 5
Pb2: 6
PbB (37 mo):
C: 3 ug/dl
Pbj: 5
Pb2: 11
PbB (1st yr):
C: ~5 jjg/d!
Pbt: 40
Pbz: 85
Pbfi (birth):
C: 5 ug/dl
Pb,: 30
Pb2: 55
PbB (12 mo):
C: 3.4 ug/dl
Age at
testing
49-
55 mo
33 mo
4-5
yr
4-5
yr
a: 12 mo
b: 16 no
Pb,: 8.6 (pulse only)
Pb2 53.4 (chronic
Pb3 55.0 (chronic
(16 mo):
C: 4
Pb,: 7.8
Pb2: 29.5
Pb3: 30.2
only)
& pulse)
Testing Non-
paradigm behavioral
(task) effects
WGTA None
(spatial
discr.
reversal
learning)
Operant None
(FI-1 min)
WGTA- None
Hamilton
search task
(find food
under 6 boxes)
Same None
(find food
under 8
boxes)
WGTA None
(discr.
reversal
learning:
a: without
overtrng;
b: with
overtrng.)
Behavioral
results
Both Pb groups
retarded in rever-
sal learning;
3 Pb2-Ss failed to
retain motor pattern
for operating MGTA
from 2 yr earlier.
Rate of acceleration
in FI pattern of
responding sig. re-
duced in Pbj + Pb2
Ss.
Pb-Ss sig. slower
to reach criterion
(examine 6 boxes
without repeats).
No sig. differences
in Pb- and C-Ss; all
Ss had equal diffi-
culty with criterion
of 8.
No differences between
Pb- and C-Ss at 12 mo;
Pbj and Pb3 (pulse
exposed) slower to
reach criterion on
reversal at 16 no;
no sig. difference
between Pb2- and
C-Ss.
-------
TABLE 12-5. (continued)
CO
o
Lead exposure
Reference
Rice
& Willes
(1979)
et al.
(1979)
Rice (1984)
Rice (198Sa)
cone.
Species (Bediua)
Macaca 500
fascicu- ug/kg
TarTs b.w. Pb
(•ilk)
Continuation of
Macaca 500 ug/kg
fascicu- b.w. Pb
laris (Milk)
Macaca 50 or
fascicu- 100 ug/kg
laris Pb («ilfc)
period
(route)
Birth -
life
(direct)
Rice & Willes
Birth -
life
(direct)
Birth -
life
(direct)
Treatment
groups
(n)
C (4)
Pb (4)
(1979)
C(4)
Pb (4)
C (7)
Pbi (8)
Pb2 (5)
Tissue lead Age at
(age measured) testing
PbB 421-
(200 d): 714 d
C: <5 ug/dl
Pb: 35-70
(400 d):
Pb: 20-50
PbB (400+ d): 2.5-
20-30 pg/dl 3 yr
PbB (peak): 3-
C: <5 ug/dl 3.5 yr
Pb: 55.3
(steady state):
Pb: 32.8
PbB (peak): 3-4
C: 3.5 ug/dl yr
Pbi; 15.4
Pb2: 25.4
(steady state):
C: 2.9
Pb^ 10.9
Pb2: 13.1
Testing Non-
paradign behavioral
(task) effects
WGTA None
(form
discrioi.
reversal)
Operant None
(multiple
FI-TO)
Operant None
(delayed
•atching to
sanple for:
1) color
2) position)
Operant None
(1) for* (incl. b.w.
discr. and blood
reversal; chem'stry)
2) color
discr. reversal;
3) fom
discr. reversal,
color irrelevant)
Behavioral
results
Pb-Ss slower
to learn successive
reversals.
Pb-Ss responded
at higher rates,
had shorter IRTs,
and tended to
respond store during
tine-out
(unrewarded).
Pb-Ss performed both
lasts sig. worse
than C-Ss, although
no difference in ac-
quisition or at 0
delay. Pb-Ss Bade
perseveratTve errors
on color Hatching
task.
Pbx-Ss sig. worse
than C-Ss on last 4
of 15 reversals for
all 3 tasks, i.e. ,
did not improve as
•uch as C-Ss; Pb2-Ss
sig. worse than C-5s
on all reversals.
-------
TABLE 12-5. (continued)
Reference
Rice (19856)
Gilbert
(1985)
Species
Nacaca
fa^cicu-
laris
fascicu-
laris
Lead exi
cone.
(medium)
2000
ug/kg
b.w. Pb
(•ilk)
oosure
period
(route)
Birth -
life
(direct)
Treatment
groups
(n)
C (6)
Pb (6)
Tissue lead
(age Measured)
PbB (peak):
C: 3.1 M9/dl
Pb: 115.0
(steady state):
C: 3.5
Pb: 33.0
Age at
testing
a: 0-9
•°;
b: 3-4
yr
Testing
paradigm
(task)
Operant
(a: FI
2 Bin
or FR 10-40;
b: Milt
FI-FR)
(ORL)
Non-
behavioral
effects
None
Behavioral
results
At 0-9 »o, Pb-Ss
paused sig. longer;
at 3-4 yr, Pb-Ss had
sig. shorter IRTs,
higher response
rate and greater
variability of re-
sponse rate.
learning to respond
at low rate; also
Abbreviations:
b.w. body weight
C control group
DRL differential reinforcement of low response rates
FI fixed interval
FR fixed ratio
IRT inter response tinte
Pb lead-exposed group (subscript indicates exposure level or other experimental condition)
Pb(Ac)2 lead acetate
PbB blood lead
S subject
WGTA Wisconsin general testing apparatus
Corrected annual averages obtained from Bushel 1 (1978).
sig. greater
session-to-session
re
i— »
co
i— »
Winneke Hacaca
et al. mulatta
(1S83);
Lilienthal
et al.
(1983)
Zook et al . Hacaca
(1980) iuTitta
350 or
600 ppm
Pb(Ac)2
(food)
1) 300
•3/kfl
or
2) 100
•g/kg Pb
(paint)
Preconception
-5 MO post-
natal (via
•other and
direct)
1) 10-23 wk
fro* age
6-8 *o or
2) 43-113 d
from age
5-12 d
(direct)
C (6)
Pb, (5)
Pb2 (6)
G! (3)c
Pbt (41
C2 (2)C
Pb2 (4)
PbB (post-test): ?
C: 9.6 ug/dl
Pb,: 51.7
Pb2: 71.4
PbB (tern.): 1) 15-16 K>
C: 12 M9/dl 2) 6 mo
Pbj: 470
Pb2: 96
WGTA ?
(series of
36 di scrim.
problems)
WGTA Various
(series of clinical
10 stimulus signs
discria.
problems)
variability in per-
formance during
terminal sessions.
Sig. dose-related
deficits in learning
set formation in
both Pb groups.
No sig. difference
in Mean number of
errors.
age-
atched controls.
-------
selected for the great majority of the behavioral studies, despite concerns that have re-
peatedly been expressed concerning the appropriateness of this species as a subject for be-
havioral investigation (e.g., Lockard, 1968, 1971; Zeigler, 1973).
A number of studies have reported alterations in learning task performances by rats with
blood lead levels below 30 ug/dl. The lowest exposure level to be significantly associated
with a behavioral effect was reported by Bushnell and Levin (1983), who exposed rats from PND
21 (postweaning) to a drinking water solution of 10 ppm lead for 35 days. Although blood lead
concentrations were not measured, brain lead levels at PND 57 (the day following termination
of lead exposure) averaged 0.05 pg/g. By comparison with other studies in which lead levels
in blood as well as brain were determined at a similar age (Collins et al., 1984; Grant et
al. , 1980; Bull et al., 1979), it would appear that the animals in question probably had
maximum blood lead levels under 20 ug/dl.
The behavior assessed by Bushnell and Levin (1983)--spontaneous alternation in a radial
arm maze—could be described as a form of natural or unrewarded learning, since there was no
experimenter-imposed contingency of reinforcement for alternating between different arms of
the maze before reentering a previously selected arm. Other testing paradigms have also re-
vealed behavioral alterations in subjects exposed to quite low levels of lead. For example,
Cory-Siechta et al. (1985) reported significant effects in rats exposed postweaning to a
25-ppm lead acetate solution for their drinking water. Exposure continued throughout the
course of the experiment, with blood lead levels stabilizing at 15-20 ug/dl by PND 99 (the
first point of measurement), by which time the behavioral effects were already evident. In
this case, the outcome was a significantly higher response rate in the lead-exposed animals on
a fixed-interval operant schedule of food reinforcement. Consistent with this finding, the
interval between bar-press responses was also significantly shorter in the lead-exposed rats.
Cory-Slechta and her colleagues obtained similar results at higher exposure levels in a series
of earlier studies (Cory-Slechta and Thompson, 1979; Cory-Slechta et al., 1981, 1983), even
when the operant schedule or contingency for reinforcement was rather different. For example,
in the experiment by Cory-Slechta et al. (1981), a bar-press of a certain minimum duration was
required before the rats could be rewarded. Subjects exposed to 100 or 300 ppm lead acetate
solutions for drinking water were impaired in their ability to meet this response requirement.
A tendency to respond more rapidly (higher response rate, shorter inter-response times,
shorter response latencies) or to respond even when inappropriate (when no reward is provided
for responses or when reward is specifically withheld for responding) has been reported in
quite a few other studies of lead-exposed rats (Alfano and Petit, 1985; Angell and Weiss,
1982; Cory-Slechta and Thompson, 1979; Cory-Slechta etal., 1983; Oietz etal,, 1978;
Gross-Selbeck and Gross Selbeck, 1981; Hastings et al., 1984; Nation et al. , 1982; Overmann,
12-132
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1977; Padich and Zenick, 1977; Rosen et al., 1985; Taylor et al., 1982; Winneke et al., 1982b;
Zem'ck et al., 1978). In many of these investigations the lead exposure levels were rather
low, resulting in blood lead concentrations under 30 ug/dl at the time of assessment (although
peak levels may have been considerably higher).
Additional forms of impairment have been reported in studies using other behavioral test-
ing paradigms. Winneke and his associates (Winneke etal., 1977, 1982b; Schlipkoter and
Winneke, 1980) employed an apparatus requiring the subjects to discriminate between stimuli of
different sizes and found that lead-exposed rats were slower to learn the discrimination or
tended to repeat errors more than control subjects. In these studies, exposure occurred jn
utero as well as via the dam's milk and directly through the subjects' drinking water post-
weaning. Blood lead levels around PND 16 were less than 30 |jg/dl in the study of Winneke
et al. (1977). A number of other reports have also noted impaired discrimination acquisition
or performance in various testing paradigms with rats (Booze et al., 1983; Geist and Mattes,
1979; Hastings etal., 1979; Kowalski etal., 1982; McLean etal., 1982; Overmann, 1977;
Penzien et al., 1982; Zenick et al., 1978).
Nonhuman primates have been studied in several studies of the effects of lead on learning
ability (Table 12-5). For the most part, these studies have exposed monkeys directly to lead
from birth and then analyzed the subjects' ability to discriminate stimuli differentially
associated with rewards. A number of these studies were conducted by Bowman and his col-
leagues (Bushnell and Bowman, 1979a,b; Levin and Bowman, 1983; Laughlin et al., 1983; Mele
et al., 1984). Using a variety of tasks and different groups of subjects (as well as the same
subjects followed for several months or years after exposure terminated), these investigators
have consistently found evidence of impaired learning ability in monkeys, even after the sub-
jects' blood lead levels had dropped to control values, i.e., ~5 |jg/dl (see Section 12.4.3.1.5
for further discussion on the persistence of neonatal exposure effects). One type of test
that has been frequently used to detect lead-induced impairment in primates is the discrimina-
tion reversal task. Discrimination reversal tasks require the subject to correctly respond to
one of two stimuli associated with reward and then, once that task has been mastered, to make
the reverse discrimination, i.e., respond only to the cue formerly unpaired with reward.
Greater difficulty in learning such reversals by lead-exposed monkeys has been shown repeated-
ly by Bowman and his colleagues.
The above findings have been generally confirmed and extended by Rice and her colleagues
(Rice, 1984, 1985a,b; Rice and Willes, 1979; Rice and Gilbert, 1985; Rice etal., 1979).
Although Rice's studies used operant conditioning tasks to a greater extent than Bowman's
studies, impaired learning ability was consistently demonstrated, even in some cases where the
monkeys' peak blood lead levels reached only 15 |jg/dl and steady state levels were only 11
ug/dl. Rice (1985a) particularly noted the consistency of her results with Bushnell and
12-133
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Bowman's (1979a,b) finding of impaired ability to learn discrimination reversal tasks. Simi-
lar results were also obtained with rats by Driscoll and Stegner (1976), but not by Hastings
et al. (1984) or Rabe et al. (1985). In addition, a relatively high degree of response varia-
bility was found in Rice's lead-treated monkeys as was found in lead-treated rats (Cory-
Slechta et al., 1985; Cory-Slechta et al. , 1981, 1983; Cory-Slechta and Thompson, 1979; Dietz
et al., 1978).
Another finding from Rice's studies that is consistent with the results of other studies
is the tendency of lead-treated subjects to respond excessively or inappropriately. For
example, lead-exposed monkeys tended to respond more than control subjects during "time-outs"
in operant schedules when responses were unrewarded (Rice and Willes, 1979). They also tended
to have higher response rates and shorter interresponse times on fixed-interval operant sched-
ules (Rice, 1985b). Where the schedule of reinforcement required a low rate of responding
before reward could be delivered, the lead-treated subjects were significantly slower than
controls to learn the appropriate pattern of responding (Rice and Gilbert, 1985). Such sub-
jects also made more perseverative errors on operant "matching-to-sample" tasks that required
them to direct their responses according to stimulus colors (Rice, 1984).
These findings bear striking resemblence to the results of several studies of lead-
exposed rats which, as mentioned above, tended to respond excessively or more rapidly than
controls or than conditions of the experiment would have otherwise produced. Such tendencies
have been characterized as "hyper-reactivity" by some investigators (e.g., Winneke et al.,
1982b). However, this concept (not to be confused with hyperactivity per se) is only descrip-
tive, not explanatory. Speculation about the neural mechanisms responsible for such behavior
has tended to focus on the hippocampus, because of the behavioral similarities with animals
having experimental lesions of the hippocampus (Petit and Alfano, 1979; Petit et al., 1983)
(see also Sections 12.4.3.2.1 and 12.4.3.5). It should be noted that, at sufficiently high
exposure levels, increased response tendencies give way to decreased responding (e.g., Cory-
Slechta and Thompson, 1979; Angel 1 and Weiss, 1982). Cory-Slechta et al. (1983) have argued
that this curvilinear dose-response relationship may be due at least in part to differences in
the time required for response rates to reach their maximum as a function of different expo-
sure levels. In their study, rats exposed to higher concentrations of lead took longer to
reach their peak response rate; consequently, assessing performance earlier would make the re-
sponding of the higher lead exposure group appear depressed, while responding of a lower expo-
sure group would appear to be elevated relative to controls (Cory-Slechta et al., 1983). Of
course, at sufficiently toxic doses, responding obviously declines if the subjects are no
longer able to perform the necessary motor responses.
12-134
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It seems clear from the above studies that alterations in the behavior of rats and
monkeys occur as a consequence of chronic exposure to relatively low levels of dietary lead.
In a number of instances (e.g., Mele et al., 1984; Bushnell and Bowman, 1979b) these perturba-
tions were evident even after blood lead concentrations had returned to nearly normal levels,
although earlier exposure had probably been much higher. One study reported learning distur-
bances in monkeys whose average steady-state blood lead level was around 11 pg/dl and whose
peak level reached only about 15 ug/dl (Rice, 1985a). A number of studies with rats found
evidence of behavioral deficits at blood lead levels below 30 ug/dl, and in at least one case
the blood lead level probably did not exceed 20 ug/dl.
12.4.3.1.4 Effects of lead on socjal behavior. The social behavior and organization of even
phylogenetically closely related species may be widely divergent. For this and other reasons,
there is little or no basis to assume that, for example, aggressiveness in a lead-treated
rhesus monkey provides a model of aggressiveness in a lead-exposed human child. However,
there are other compelling grounds for including animal social behavior in the present review.
As in the case of nonsocial behavior patterns, characteristics of an animal's interactions
with conspecifics may reflect neurological (especially CNS) impairment due to toxic exposure.
Also, certain aspects of animal social behavior have evolved for the very purpose (in a non-
teleological sense) of indicating an individual's physiological state or condition (Davis,
1982). Such behavior could potentially provide a sensitive and convenient indicator of toxi-
cological impairment.
Two early reports (Silbergeld and Goldberg, 1973; Sauerhoff and Michael son, 1973) sug-
gested that lead exposure produced increased aggressiveness in rodents. Neither report, how-
ever, attempted to quantify these observations of increased aggression. Later, Hastings et
al. (1977) examined aggressive behavior in rats that had been exposed to lead via their dams'
milk. Solutions containing 0, 0.01, or 0.05 percent lead as lead acetate constituted the dams'
drinking water from parturition to weaning at PND 21, at which time exposure was terminated.
This lead treatment produced no change in growth of the pups. Individual pairs of male off-
spring (from the same treatment groups) were tested at PND 60 for shock-elicited aggression.
Both lead-exposed groups (average blood lead levels of 5 and 9 pg/dl and brain lead levels of
8 and 14 ug/lOOg) showed significantly less aggressive behavior than the control group. There
were no significant differences among the groups in the flinch/jump thresholds for shock,
which suggests that the differences seen in shock-elicited aggression were not caused by dif-
ferences in sensitivity to shock.
A study by Drew et al. (1979) utilized apomorphine to induce aggressive behavior in 90-
day-old rats and found that earlier lead exposure attenuated the drug-induced aggressiveness.
12-135
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Lead exposure occurred between birth and weaning primarily through the dams' milk or through
food containing 0.05 percent lead as lead acetate. No blood or tissue concentrations of lead
were measured. There were no significant differences in the weights of the lead-treated and
control animals at PND 10, 20, 30, or 90.
Using laboratory mice exposed as adults, Ogilvie and Martin (1982) also observed reduced
levels of aggressive behavior. Since the same subjects showed no differences in vitality or
open field activity measures, the reduction in aggressiveness did not appear to be due to a
general effect of lead on motor activity. Blood lead levels were estimated from similarly
treated groups to be approximately 160 ug/dl after 2 weeks of exposure and 101 ug/dl after
4 weeks of exposure.
Cutler (1977) used ethological methods to assess the effects of lead exposure on social
behavior in laboratory mice. Subjects were exposed from birth (via their dams' milk) and
post-weaning to a 0.05 percent solution of lead as lead acetate (average brain lead concentra-
tions were 2.45 nmol/g for controls and 4.38 nmol/g for experimental subjects). At 8 weeks of
age social encounters between subjects from the same treatment group were analyzed in terms of
a number of specified, identifiable behavioral and postural elements. The frequency and dura-
tion of certain social and sexual investigative behavior patterns were significantly lower in
lead-treated mice of both sexes than in controls. Lead-exposed males also showed significant-
ly reduced agonistic behavior compared with controls. Overall activity levels (nonsocial as
well as social behavior) were not affected by the lead treatment. Average body weights did
not differ for the experimental and control subjects at weaning or at the time of testing.
A more recent study by Cutler and coworkers (Donald et al., 1981) used a similar
paradigm of exposure and behavioral evaluation, except that exposure occurred either only pre-
natal ly or postnatal ly and testing occurred at two times, 3-4 and 14-16 weeks of age. Sta-
tistically significant effects were found only for the postnatal exposure group. Although
total activity in postnatally exposed mice did not differ from that of controls at either age
of testing, the incidence of various social activities did differ significantly. As juveniles
(3-4 weeks old), lead-treated males (and to some extent, females) showed decreased social in-
vestigation of a same-sex conspecific. This finding seems to be consistent with Cutler's
(1977) earlier observations made at 8 weeks of age. Aggressive behavior, however, was almost
nonexistent in both control and lead-treated subjects in the later study, and so could not be
compared meaningfully. Although the authors do not comment on this aspect of their study, it
seems likely that differences in the strains of laboratory mice used as subjects could well
have been responsible for the lack of aggressive behavior in the Donald et al. (1981) study
(see, e.g., Adams and Boice, 1981). Later testing at 14-16 weeks revealed that lead-exposed
female subjects engaged in significantly more investigative behavior of a social or sexual
12-136
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nature than did control subjects, while males still showed significant reductions in such
behavior when encountering another mouse of the same sex. This apparent disparity between
male and female mice is one of relatively few reports of gender differences in sensitivity to
lead's effects on the nervous system (cf. Cutler, 1977; Verlangieri, 1979). In this case,
Donald et al. (1981) hypothesized that the disparity might have been due to differences in
brain lead concentrations: 74.7 (jmol/kg in males versus 191.6 umol/kg in females (blood lead
concentrations were not measured).
The social behavior of rhesus monkeys has also been evaluated as a function of early lead
exposure. A study by Allen et al. (1974) reported persistent perturbations in various aspects
of the social behavior of lead-exposed infant and juvenile monkeys, including increased cling-
ing, reduced social interaction, and increased vocalization. However, exposure conditions
varied considerably in the course of this study, with overt toxicity being evident as blood
lead levels at times ranged higher than 500 ug/dl.
A more recent study consisting of four experiments (Bushnell and Bowman, 1979c) also
examined social behavior in infant rhesus monkeys, but under more systematically varied expo-
sure conditions. In experiments 1 and 2, daily ingestion of lead acetate during the first
year of life resulted in blood lead levels of 30-100 pg/dl, with consequent suppression of
play activity, increased clinging, and greater disruption of social behavior when the play
environment was altered. Experiment 3, a comparison of chronic and acute lead exposure (the
latter resulting in a peak blood lead concentration of 250-300 ug/dl during weeks 6-7 of
life), revealed little effect of acute exposure except in the disruption that occurred when
the play environment was altered. Otherwise, only the chronically exposed subjects differed
significantly from controls in various categories of social behavior. Experiment 4 of the
study showed that prenatal exposure alone, with blood lead concentrations of exposed infants
ranging between 33 and 98 ug/dl at birth, produced no detectable behavioral effects under the
same procedures of evaluation. Overall, neither aggressiveness nor dominance was clearly
affected by lead exposure.
Another aspect of social behavior--interaction between mothers and their offspring—was
examined in lead-exposed rats by Zenick et al. (1979). Dams chronically received up to 400
mg/kg lead acetate in their drinking water on a restricted daily schedule (blood lead concen-
trations averaged 96.14 ± 16.54 ug/dl in the high-exposure group at day 1 of gestation). Dams
and their litters were videotaped on PND 1-11, and the occurrence of certain behavior patterns
(e.g., lying with majority of pups, lying away from pups, feeding) was tabulated by the exper-
imenters. In addition, dams were tested for their propensity to retrieve pups removed from
the nest. Neither analysis revealed significant effects of lead exposure on the behavior of
the dams. However, restricted access to drinking water (whether lead-treated or not) appeared
to confound the measures of maternal behavior.
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A more recent investigation of maternal behavior and offspring development in rats ex-
posed via their food revealed significant lead-related alterations in the behavioral inter-
actions between pups and their dams (Barrett and Livesey, 1983). Pups whose blood lead levels
ranged from 20 to 60 ug/dl at weaning were slower to leave the nest area to find the dam for
suckling or to climb into food hoppers for solid food. The lead-exposed dams, with blood lead
values of 30-60 ug/dl at weaning, in turn spent more time in the nest than control dams.
These findings are consistent with other observations of retarded pup development and in-
creased retrieval of pups to the nest by dams exposed to low levels of lead (Davis, 1982). As
Barrett and Livesey (1983) note, the net effect of this altered motor-infant interaction is
difficult to predict. While extra maternal care could help compensate for slowed development
caused by lead, it could also exacerbate the situation by depriving the pups of the outside
stimulation needed for normal development (Levitsky et al., 1975).
The above studies suggest that animal social behavior or behavioral interactions may be
altered in various ways by exposure to lead. Aggressive behavior in particular is, if any-
thing, reduced in laboratory animals as a result of exposure to lead. Certain other aspects
of social behavior in laboratory mice, namely components of sexual interaction and social in-
vestigation, also appear to be reduced in lead-treated subjects, although there may be gender
differences in this regard following chronic post-maturational exposure. In additon, young
rhesus monkeys appear to be sensitive to the disruptive effects of lead on various aspects of
social behavior. These alterations in social behavior in several mammalian species are indi-
cative of altered neural functioning as a consequence of lead exposure.
12.4.3.1.5 Persistence of neonatal exposure effects. The specific question of persisting,
long-term consequences of lead exposure on the developing organism has been addressed in a
number of studies by carrying out behavioral testing some time after the termination of lead
exposure. For example, such evidence of long-term effects has been reported for rhesus
monkeys by Bushnell and Bowman (1979b). Their subjects were fed lead acetate so as to main-
tain blood lead levels of either 50 ± 10 (low-lead) or 80 ± 10 ug/dl (high-lead) throughout
the first year of life (actual means and standard errors for the year were reported as 31.71 ±
2.75 and 65.17 ± 6.28 ug/dl). Lead treatment was terminated at 12 months of age, after which
blood lead levels declined to around 5-6 ug/dl at 56 months. At 49 months of age the subjects
were re-introduced to a discrimination reversal training procedure using new discriminative
stimuli. Despite their extensive experience with the apparatus (Wisconsin General Test
Apparatus) during the first two years of life, most of the high-lead subjects failed to retain
the simple motor pattern (pushing aside a small wooden block) required to operate the ap-
paratus. Remedial training largely corrected this deficit. However, both high- and low-lead
groups required significantly more trials than the control group (p <0.05) to reach criterion
12-138
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performance levels. This difference was found only on the first discrimination task and nine
reversals of it. Successive discrimination problems showed no differential performance ef-
fects, which indicates that with continued training the lead-treated subjects were able to
achieve the same level of performance as controls.
Other studies with monkeys have also shown behavioral alterations some time after blood
lead concentrations have returned to essentially normal levels (Laughlin et al., 1983; Levin
and Bowman, 1983; Mele et al., 1984). Some evidence suggests that rats may show similar
effects (e.g., Angell and Weiss, 1982; Gross-Selbeck and Gross-Selbeck, 1981), but other
evidence implies that behavioral effects eventually disappear after lead exposure ends (e.g.,
Flynn et al., 1979; Hastings et al., 1977, 1984; Padich and Zenick, 1977; Rosen et al., 1985;
Schlipkb'ter and Winneke, 1980). Even if some behavioral changes are reversible, it does not
follow, of course, that all behavioral effects of early lead exposure are reversible. Most
likely, neurotoxic outcomes differ in their persistence, and these differences account for any
apparent inconsistency in the above findings.
12.4.3.2 Morphological Effects
12.4.3.2.1 In vivo studies. Recent key findings on the morphological effects of i£ vivo lead
exposure on the nervous system are summarized in Table 12-6.* It would appear that certain
types of of glial cells are sensitive to lead exposure, as Reyners et al. (1979) found a de-
creased density of oligodendrocytes in cerebral cortex of young rats exposed from birth to 0.1
percent lead in their food. Exposures to higher concentrations (0.2-0.4 percent lead salts),
especially if begun during the prenatal period (Bull et al., 1983), can reduce synaptogenesis
and retard dendritic development in the cerebral cortex (McCauley and Bull, 1978; McCauley et
al., 1979, 1982) and the hippocampus of developing rats (Campbell et al., 1982; Alfano and
Petit, 1982). Some of these effects, e.g., those on the hippocampus, appear to be transient
(Campbell et al., 1982) and may be related to lead-induced alterations in size and/or bio-
availability of sub-cellular zinc pools (Sato et al., 1984). Interestingly, an apparent com-
pensatory hypertrophy of both neurons and neuropil appears in certain areas of the hippocampus
of 90-day old rats who were exposed perinatally to lead (Kawamoto et al., 1984).
"Concentrations of lead reported in the following sections are given as percent lead salt.
For comparison with exposure concentrations discussed in other sections of this document,
multiply by 10,000 to obtain value in parts per million (ppm). Example: 1% = 10,000 ppm.
12-139
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TABLE 12-6. SUMMARY OF KEY STUDIES OF MORPHOLOGICAL EFFECTS OF IN VIVO LEAD EXPOSURE*
Species
Exposure protocol
Peak blood
lead level
Observed
effect
Reference
Young rats
0.1% Pb2* in chow
PND 0-90
0.1% Pb(Ac)2 in
dams' drinking
water PND 0-60
0.1X Pb(Ac)2 in
dams' drinking
water PND 0-32
0.2% PbCl2 in dams'
drinking water from
gestation thru PND 20
0.2% Pb(Ac)2 1n
dams' drinking
water PND 0-25
0.4% PbC03 in
dams' drinking
water PND 0-30
0.5% Pb(Ac)2 in
dams' drinking
water PND 0-21
1% PbCOa in chow
PND 0-60
4% PbC03 in dams'
chow PND 0-28
80 ug/dl
(at birth)
300-400 ug/dl
(PND 26)
385 ug/dl
(PND 21)
258 Mg/dl
(PND 28)
Decreased density of
oligodendrocytes 1n cerebral
cortex
Focal necrosis of photoreceptor
cells and cells In Inner
nuclear layer of retina
Significant Inhibition In
myelin deposition and
maturation in whole brain
Less mature synaptlc profile
in cerebral cortex at PNO 15
30% reduction in synaptlc
density in cerebral cortex
at PND 15 (returned to normal
at PND 21)
15-30% reduction in
synaptic profiles in
hippocampus
Retardation in temporal
sequence of hippocanpal
dendritic development
10-15% reduction in number
of axons 1n optic nerve;
skewing of fiber diameters
to smaller sizes
Retardation of cortical
synaptogenesis over and
above any nutritional
effects
13% reduction in
cortical thickness
and total brain weight;
reduction in synaptlc
density
•Abbreviations:
PND: postnatal day
Pb(Ac)2: lead acetate
PbC03: lead carbonate
Reyners et al. (1979).
Santos-Anderson et al.
(1984)
Stephens and
Gerber (1981)
McCauley and Bull
(1978); McCauley
et al. (1979)
McCauley et al. (1982)
Campbell et al. (1982)
Alfano and Petit
(1982); Petit et al.
(1983)
Tennekoon et al.
(1979)
Averill and Needleman
(1980)
Petit and
LeBoutilller (1979)
4% PbC03 in dams'
chow PND 0-25
Adult rats 4% PbC03 in chow
for 3 MOS.
4% PbC03 in chow 300 ug/dl
PND 0-150 (PND 150)
Reduction In hippocanpal
length and width; similar
reduction in afferent
projection to hippocampus
Delay in onset and peak
of Schwann cell division
and axonal regrowth in
regenerating nerves
Demyell nation of peri-
pheral nerves beginning
PND 20-35
Alfano et al. (1982)
Ohnishi and
(1981)
Dyck
Wlndebank et al.
(1980)
12-140
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Suckling rats subjected to increasing exposures of lead exhibit more pronounced effects,
such as reduction in the number and average diameter of axons in the optic nerve at 0.5 per-
cent lead acetate exposure (Tennekoon et al., 1979), a general retardation of cortical syn-
aptogenesis at 1.0 percent lead carbonate exposure (Averill and Needleman, 1980), or a reduc-
tion in cortical thickness at 4.0 percent lead carbonate exposure (Petit and LeBoutillier,
1979). This latter exposure concentration also causes a delay in the onset and peak of
Schwann cell division and axonal regrowth in regenerating peripheral nerves in chronically
exposed adult rats (Ohnishi and Dyck, 1981). In short, both neuronal and glial components of
the nervous system appear to be affected by neonatal or chronic lead exposure.
Organolead compounds have also been demonstrated to have a deleterious effect on the mor-
phological development of the nervous system. Seawright et al. (1980) administered triethyl
lead acetate (Et3Pb) by gavage to weanling (40-50 g) and "young adult" (120-150 g) rats.
Single doses of 20 mg Et3Pb/kg caused impaired balance, convulsions, paralysis, and coma in
both groups of treated animals. Peak levels in blood and brain were noted two days after ex-
posure, with extensive neuronal necrosis evident in several brain regions by three days post-
treatment. Weekly exposures to 10 mg Et3Pb/kg for 19 weeks resulted in less severe overt
signs of intoxication (from which the animals recovered) and moderate to severe loss of neu-
rons in the hippocampal region only.
12.4.3.2.2 In vitro studies. Bjorklund et al. (1980) placed tissue grafts of developing ner-
vous tissue in the anterior eye chambers of adult rats. When the host animals were given 1 or
2 percent lead acetate in their drinking water, the growths of substantia nigral and hippocam-
pal, but not cerebellar, grafts were retarded. Grafts of the developing cerebral cortex in
host animals receiving 2 percent lead exhibited a permanent 50 percent reduction in size
(volume), whereas 1 percent lead produced a slight increase in size in this tissue type. The
authors felt that this anomalous result might be explained by a hyperplasia of one particular
cell type at lower concentrations of lead exposure.
Organolead compounds have also been demonstrated to affect neuronal growth (Grundt et
al., 1981). Cultured cells from embryonic chick brain were exposed to 3.16 uM triethyllead
chloride in the incubation medium for 48 hr, resulting in a 50 percent reduction in the number
of cells exhibiting processes. There was no observed effect on glial morphology.
Other investigations have focused on morphological aspects of the blood-brain barrier and
its possible disruption by lead intoxication (Kolber et al., 1980). Capillary endothelial
cells isolated from rat cerebral cortex and exposed to 100 uM lead acetate iji vitro
(Silbergeld et al., 1980b) were examined by electron microscopy and X-ray microprobe analysis.
Lead deposits were found to be sequestered preferentially in the mitochondria of these cells
in much the same manner as calcium. This affinity may be the basis for lead-induced disrup-
tion of transepithelial transport of Ca2 and other ions.
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12.4.3.3 Electrophysiological Effects.
12.4.3.3.1 In vivo studies. Recent key findings on the electrophysiological effects of ir\
vivo lead exposure are summarized below in Table 12-7. The visual system appears to be par-
ticularly susceptible to perturbation by neonatal lead exposure. Suckling rats whose dams
were given drinking water containing 0.2 percent lead acetate had significant alterations in
their visual evoked responses (VERs) and decreased visual acuity at PND 21, at which time
their blood lead levels were 65 ug/dl (Cooper et al., 1980; Fox et al., 1977; Impelman et al. ,
1982; Fox and Wright, 1982; Winneke, 1980). Both of these observations are indicative of
depressed conduction velocities in the visual pathways. These same exposure levels also in-
creased the severity of the maximal electroshock seizure (MES) response in weanling rats who
exhibited blood lead levels of 90 ug/dl (Fox et al., 1978, 1979). The authors speculated that
neonatal lead exposure acts to increase the ratio of excitatory to inhibitory systems in the
developing cerebrospinal axis. Such exposure can also lead to lasting effects on the adult
nervous system, as indicated by persistent decreases in visual acuity and spatial resolution
in 90-day old rats exposed only from birth to weaning to 0.2 percent lead acetate (Fox et al.,
1982). A 38-percent decrease in the number of cholinergic receptors in the visual cortex of
adult rats treated in this manner (Costa and Fox, 1983) may represent the morphological basis
for this finding.
The adult nervous system is also vulnerable to lead-induced perturbation at low levels of
exposure. For example, Hietanen et al. (1980) found that chronic exposure of adult rabbits to
0.2 percent lead acetate in drinking water resulted in an 85 percent inhibition of motor con-
duction velocity in the sciatic nerve; adult rabbits fed 165 mg lead carbonate per day for 5
days (Kim et al., 1980) showed a 75 percent increase in Ca2 retention time in incubated brain
slices, indicating that lead inhibits the mediated efflux of Ca2 .
12.4.3.3.2 In vitro studies. Palmer et al. (1981) and Olson et al. (1981) looked at intra-
ocular grafts of cerebellar tissue from 14- to 15-day-old rats in host animals treated for 2
months with drinking water containing 1 percent lead acetate, followed by plain water for 4-5
months. They found no alterations in total growth or morphology of cerebellar grafts in
treated versus control hosts, yet the Purkinje neurons in the lead-exposed grafts had almost
no spontaneous activity. Host cerebellar neurons, on the other hand, and both host and graft
neurons in control animals, all exhibited significant levels of spontaneous activity. It
should be noted that when these investigators looked at the effects of lead on intraocular
grafts of other areas of fetal rat brain, i.e., substantia nigra, hippocampus, and parietal
cortex, they found significant delays in the growth of these grafts (Olson et al. , 1984).
Furthermore, attempts by this group to replicate their findings ui vivo by using neonatal rats
exposed from gestation to PND 20 to 0.5 percent lead acetate in drinking water have been un-
successful (Palmer et al., 1984).
12-142
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TABLE 12-7. SUMMARY OF KEY STUDIES OF ELECTROPHYSIOLOGICAL EFFECTS OF IN VIVO LEAD EXPOSURE*
Species
Exposure protocol
Peak blood
lead level
Observed
effect
Reference
Suckling rat
I
1—"
to
Young rhesus
monkeys
Adult rabbit
0.2% Pb(Ac)? in
dams' drinking water
PND 0-20
0.2% Pb(Ac)? in
dams' drinking water
PND 0-21
Pb(Ac)2 solutions
in food
PNO 0-365
0.2% Pb(Ac)2 in
drinking water for
4 weeks
90
(PND 20)
65
(PND 21)
300
(PND 60)
85 jjg/dl
More rapid appearance
and increased severity of
MES response
1) Increased latencies and
decreased amplitudes of
primary and secondary
components of VER;
2) decreased conduction
velocities in visual
pathways;
3) 25-50% decrease in
scotopic visual acuity
4) persistent decreases
in visual acuity and
spatial resolution at
PND 90
Severe impairment
of discrimination
accuracy; loss of
scotopic function
85% reduction in motor
conduction velocity of
sciatic nerve
Fox et al.
(1978, 1979)
Fox et al.
(1977);
Impelman et al.
(1982);
Cooper et al.
(1980);
Winneke (1980);
Fox and Wright
(1982);
Fox et al.
(1982)
Bushnell et al.
(1977)
Hietanen et al.
(1980)
*Abbreviations:
PNO: postnatal day
Pb(Ac)2: lead acetate
MES: maximal electroshock seizure
VER: visual evoked response
-------
Taylor et al. (1978) recorded extracellularly from cerebellar Purkinje cells in adult
rats both ui situ and in intraocular grafts in an effort to determine what effect lead had on
the norepinephrine (NE)-induced inhibition of Purkinje cell spontaneous discharge. Applica-
tion of exogenous NE to both rn situ and i_n oculo cerebellum produced 61 and 49 percent inhi-
bitions of spontaneous activity, respectively. The presence of 5-10 uM lead reduced this in-
hibition to 28 and 13 percent, respectively. This "disinhibition" was specific for NE, as
responses to both cholinergic and parallel fiber stimulation in the same tissue remained the
same. Furthermore, application of lead itself did not affect spontaneous activity, but did
inhibit adenylate cyclase activity in cerebellar homogenates at the same concentration re-
quired to disinhibit the NE-induced reduction of spontaneous activity (3-5 uM).
Fox and Sillman (1979) and Sillman et al. (1982) looked at receptor potentials in the
isolated, perfused bullfrog retina and found that additions of lead chloride caused a rever-
sible, concentration-dependent depression of rod (but not cone) receptor potentials. Concen-
trations as low as 1 uM produced an average 5 percent depression, while 25-60 uM produced an
average 34 percent depression.
Evidence that lead does indeed resemble other divalent cations, in that it appears to
interfere with chemically-mediated synaptic transmission, has also been obtained in studies of
peripheral nerve function. For example, lead is capable of blocking neural transmission at
peripheral adrenergic synapses (Cooper and Steinberg, 1977). Measurements of the contraction
force of the rabbit saphenous artery following stimulation of the sympathetic nerve endings
indicated that lead blocks muscle contraction by an effect on the nerve terminals rather than
by an effect on the muscle. Since the response recovered when the Ca2 concentration was in-
creased in the bathing solution, it was concluded that lead does not deplete transmitter
stores in the nerve terminals, but more likely blocks NE release (Cooper and Steinberg, 1977;
Pickett and Bornstein, 1984; Kober and Cooper, 1976).
It has also been demonstrated that lead depresses synaptic transmission at the peripheral
neuromuscular junction by impairing acetylcholine (ACh) release from presynaptic terminals
(Kostial and Vouk, 1957; Manalis and Cooper, 1973; Cooper and Manalis, 1974). This depression
of neurotransmitter release evoked by nerve stimulation is accompanied by an increase in the
spontaneous release of ACh, as evidenced by the increased frequency of spontaneous miniature
endplate potentials (MEPPs) (Atchison and Narahashi, 1984; Kolton and Yaari (1982) and Manalis
et al. (1984) found that this increase in MEPPs in the frog nerve/muscle preparation could be
induced by lead concentrations as low as 5 uM and is probably due to competitive inhibition of
Ca2+ binding (Cooper et al., 1984).
The effects of lead on neurotransmission within the central nervous system have also been
studied. For example, investigation of the ui vitro effects of lead on Ca2 binding on cau-
date synaptosomes was carried out by Silbergeld and Adler (1978) . They determined that 50 uM
12-144
-------
lead caused an 8-fold increase in 45Ca2+ binding and that in both control and lead-treated
preparations the addition of ATP increased binding, while ruthenium red and Ca2+ decreased it.
Further findings in this series of experiments demonstrated that lead inhibits the Na+-
stimulated loss of Ca2 by mitochondria and that blockade of dopamine (DA) uptake by 5 pM
benztropine reversed the lead-stimulated increase in Ca2"1" uptake by synaptosomes. The authors
concluded that lead affects the normal mechanisms of Ca2"1" binding and uptake, perhaps by che-
lating with DA in order to enter the nerve terminal. By inhibiting the release of Ca2+ bound
to mitochondria there, lead essentially causes an increase in the Ca2 concentration gradient
across the nerve terminal membrane. As a result, more Ca2+ would be expected to enter the
nerve terminal during depolarization, thus effectively increasing synaptic neurotransmission
at dopaminergic terminals without altering neuronal firing rates.
12.4.3.4 Biochemical Alterations. The majority of previous investigations of biochemical
alterations in the nervous system following exposure to lead have focused on perturbations of
various neurotransmitter systems, probably because of the documentation extant on the neuro-
physiological and behavioral roles played by these transmitters. Recently, however, somewhat
more attention has been centered on the impact of lead exposure on energy metabolism and other
cellular homeostatic mechanisms such as protein synthesis and glucose transport. A signifi-
cant portion of this work has, however, been conducted \r\ vitro.
12.4.3.4.1 In vivo studies. Recent key findings on the biochemical effects of in vivo expo-
sure are summarized in Table 12-8. Although the majority of recent work has continued to
focus on neurotransmitter function, it appears that the mechanisms of energy metabolism are
also particularly vulnerable to perturbation by lead exposure. McCauley, Bull, and coworkers
have demonstrated that exposure of prenatal rats to 0.02 percent lead chloride in their dams'
drinking water leads to a marked reduction in cytochrome content in cerebral cortex, as well
as a possible uncoupling of energy metabolism. Although the reduction in cytochrome content
is transient and disappears by PND 30, it occurs at blood lead levels as low as 36 ug/dl
(McCauley and Bull, 1978; Bull et al., 1979); delays in the development of energy metabolism
may be seen as late as PND 50 (Bull, 1983). [See Section 12.2.1.3 for a discussion of lead
effects on mitochondrial function.]
There does not appear to be a selective vulnerability of any particular neurotransmitter
system to the effects of lead exposure. Pathways utilizing dopamine (DA), norepinephrine
(NE), serotonin (5-HT), y-aminobutyric acid (GABA), and acetylcholine (ACh) as neuro-
transmitters are all reported to be affected in neonatal animals at lead-exposure con-
centrations of 0.2-2.0 percent lead salts in dams' drinking water (see Shellenberger, 1984 for
an exhaustive review of this literature). Although the blood lead values reported following
exposure to the lower lead concentrations (0.2-0.25 percent lead acetate or lead chloride)
12-145
-------
TABLE 12-8. SUMMARY OF KEY STUDIES ON BIOCHEMICAL EFFECTS OF IN VIVO LEAD EXPOSURE
Subject
Suckling rat
Peak blood
Exposure protocol lead level
0.004% Pb(Ac)2 in
dams' drinking water
PND 0-35
Observed effect
Decline in synthesis and
turnover of striatal DA
Reference
Govoni et al.
(1979,
1980); Memo
et al.
(1980a>
1981)
0.02% PbCl2 in dams'
drinking water from
gestation thru PND
21
80 ug/dl 1) Transient 30% reduction
(at birth) in cytochrome content of
36 ug/dl cerebral cortex;
(PND 21) 2) possible uncoupling of
energy metabolism
3) delays in development of
energy metabolism
0.2% Pb(Ac)2 in 47 ug/dl
dams' drinking water (PND 21)
PND 0-21
0.2% Pb(Ac)2 in
dams' drinking water
PND 0-20
0.25% Pb(Ac)2 in
dams' drinking water
PND 0-35
0.25% Pb(Ac)2 in
dams' drinking water
PND 0-35
0.25% Pb(Ac)2 in
dams' drinking water
PND 0-35
0.25% Pb(Ac2) in
dams' drinking water
PND 0-56
1) 23% decrease in NE levels
of hypothalamus and
striatum;
2) increased turnover of
NE in brainstem
8% decrease in AChE
activity in cerebellum
Decline in synthesis
and turnover of striatal
DA
Increase in DA synthesis
in frontal cortex and
nuc. accumbens(10-30%
and 35-45%, respectively)
McCauley and
Bull
(1978);
McCauley
et al.
(1979);
Bull et al.
(1979);
Bull (1983)
Goldman et
al. (1980)
Gietzen and
Woolley
(1984)
Govoni et al.
(1978a)
Govoni et al.
(1979,
1980; Memo
et al.
(1980a,
1981)
1) 50% increase in DA-specific Lucchi et al.
binding to striatal (1981)
D2 receptors;
2) 33% decrease in DA-specific
binding to nuc. accumbens
D2 receptors
1) Decline in uptake of DA Missale
by striated nerve endings et al.
2) Elevated DA uptake in (1984)
nuc. accumbens
12-146
-------
TABLE 12-8. (continued)
Subject
Peak blood
Exposure protocol lead level
0.25% Pb(Ac)2 71 ug/dl
dams' drinking water (PND 56)
PND 0-56
Observed effect
27% decrease in DA-specific
binding to pituitary D2
receptors
Reference
Govoni et al.
(1984)
0.25% Pb(Ac)2 in 87 ug/dl
dams' drinking water (PND 42)
PND 0-42
0.25% Pb(Ac)2 in
dams' drinking water
PND 0-21; 0.004% or
0.25% until PND 42
0.5-1% Pb(Ac)2 in
drinking water
PND 0-60
0.25-1% Pb(Ac)2 in
drinking water
PND 0-60
75 mg Pb(Ac)2/kg
b.w./day via
gastric intubation
PND 2-14
72-91 g/dl
(PND 21)
98
(PND 15)
1) 31% increase in GABA Govoni et al,
specific binding in (1978b,
cerebellum; 53% increase 1980)
in GMP activity;
2) 36% decrease in GABA-
specific binding in striatum;
47% decrease in GMP activity
1) 12 and 34% elevation of Memo et al.
GABA binding in cerebellum (1980b)
for 0.004% and 0.25%,
respectively;
2) 20 and 45% decreases in
GABA binding in striatum for
0.04 and 0.25%, respectively
1) Increased sensitivity Silbergeld
to seizures induced et al.
by GABA blockers; (1979,
2) increase in GABA synthesis 1980a)
in cortex and striatum;
3) inhibition of GABA uptake
and release by synaptosomes
from cerebellum and basal
ganglia;
4) 70% increase in GABA-
specific binding in
cerebellum
1) 40-50% reduction of Modak et al.
whole-brain ACh by PND 21; (1978)
2) 36% reduction by PND 30
(return to normal values
by PND 60)
1) 20% decline in striatal Jason and
DA levels at PND 35; Kellogg
2) 35% decline in striatal (1981)
DA turnover by PND 35;
3) Transient depression of
DA uptake at PND 15;
4) Possible decreased DA
terminal density
12-147
-------
TABLE 12-8. (continued)
Subject
Exposure protocol
Peak blood
lead level
Observed effect
Reference
Young rat 2% Pb(Ac)2 in dams'
drinking water PND 0-21
then 0.002-0.008% until
PND 56
1) non-dose-dependent Dubas et al,
elevations of NE in (1978)
midbrain (60-90%) and
DA and 5-HT in midbrain,
striatum and hypothalamus
(15-30%);
2) non-dose-dependent depression
of NE in hypothalamus and
striatum (20-30%).
*Abbreviations:
PNO: postnatal day
Pb(Ac)2: lead acetate
PbCl2: lead chloride
NE: norepinephrine
DA: dopamine
GABA: Y~aroinobutyric acid
GMP: guanosine monophosphate
5-HT: serotonin
ACh: acetylcholine
AChE: acetylcholinesterase
b.w.: body weight
range from 47 M9/dl (Goldman et al., 1980) to 87 |jg/dl (Govoni et al., 1980), a few general
observations can be made:
(1) Synthesis, turnover, and uptake of DA and NE are depressed in the striatum, and
elevated in midbrain, frontal cortex, and nucleus accumbens. This seems to be
paralleled by concomitant increases in DA-specific binding in striatum and
decreases in DA-specific binding in nucleus accumbens, possibly involving a
specific subset (D2) of DA receptors (Lucchi et al., 1981). These findings are
probably reflective of sensitization phenomena resulting from changes in the
availability of neurotransmitter at the synapse.
(2) The findings for pathways utilizing GABA show similar parallels. Increases in
GABA synthesis in striatum are coupled with decreases in GABA-specific binding
in that region, while the converse holds true for the cerebellum. In these
cases, cyclic GMP activity mirrors the apparent changes in receptor function.
This increased sensitivity of cerebellar postsynaptic receptors (probably a
response to the lead-induced depression of presynaptic function) is likely the
basis for the finding that lead-treated animals are more susceptible to
seizures induced by GABA-blocking agents such as picrotoxin or strychnine
(Silbergeld et al., 1979).
12-148
-------
12.4.3.4.2 In vitro studies. Any alterations in the integrity of the blood-brain barrier can
have serious consequences for the nervous system, especially in the developing organism.
Kolber et al. (1980) examined glucose transport in isolated microvessels prepared from the
brains of suckling rats given 25, 100, 200, or 1000 rag lead/kg body weight daily by intra-
gastric gavage. On PND 25, they found that even the lowest dose blocked specific transport
sites for sugars and damaged the capillary endothelium. In vitro treatment of the preparation
with concentrations of lead as low as 0.1 uM produced the same effects.
Purdy et al. (1981) examined the effects in rats of varying concentrations of lead ace-
tate on the whole-brain synthesis of tetrahydrobiopterin (BH4), a cofactor for many important
enzymes, including those regulating catecholamine (e.g., DA or NE) synthesis. Concentrations
of lead as low as 0.01 uM produced a 35 percent inhibition of BH4 synthesis, while 100 uM in-
hibited the BH4 salvage enzyme, dihydropteridine reductase, by 40 percent. This would result
in a decreased conversion of phenylalanine to tyrosine and thence to DOPA (the initial steps
in dopamine synthesis), as well as decreases in the conversion of trytophan to its 5-hydroxy
form (the initial step in serotonin synthesis). These decrements, if occurring in vivo, could
not be ameliorated by increased dietary intake of BH4, as it does not cross the blood-brain
barrier.
Lead has also been found to have an inhibitory effect on mitochondrial respiration in
the cerebrum and cerebellum of immature or adult rats at concentrations greater than 50 uM
(Holtzman et al., 1978). This effect, which was equivalent in both brain regions at both
ages studied, is apparently due to an inhibition of nicotinamide adenine dinucleotide
(NAD)-linked dehydrogenases within the mitochondrial matrix. These same authors found that
this lead-induced effect, which is an energy-dependent process, could be blocked jn vitro by
addition of ruthenium red to the incubation medium (Holtzman et al., 1980b). In view of the
fact that Ca2 uptake and entry into the mitochondrial matrix is also blocked by ruthenium
red, it is possible that both lead and Ca2+ share the same binding site/carrier in brain mito-
chondria. These findings are supported by the work of Gmerek et al. (1981) on adult rat cere-
bral mitochondria, with the exception that they observed respiratory inhibition at 5 uM lead
acetate, which is a full order of magnitude lower than the Holtzman et al. (1978, 1980b)
studies. Gmerek and co-workers offer the possibility that this discrepancy may have been due
to the inadvertent presence of EDTA in the incubation medium used by Holtzman and co-workers.
Organolead compounds have also been demonstrated to have a deleterious effect on cellular
metabolism in the nervous system. For example, Grundt and Neskovic (1980) found that concen-
trations of triethyl lead chloride as low as 5-7 uM caused a 40 percent decrease in the incor-
poration of S04 or serine into myelin galacto-lipids in cerebellar slices from 2-week-old
rats. Similarly, Konat and coworkers (Konat and Clausen, 1978, 1980; Konat et al., 1979) ob-
serv.ed that 3 uM triethyl lead chloride preferentially inhibited the incorporation of leucine
12-149
-------
Into myelin proteins in brain stem and forebrain slices from 22-day-old rats. This apparent
inhibition of myelin protein synthesis was twofold greater than that observed for total pro-
tein synthesis (approximately 10 versus 20 percent, respectively). In addition, acute intoxi-
cation of these animals by i.p. injection of triethyl lead chloride at 8 mg/kg produced equi-
valent results accompanied by a 30 percent reduction in total forebrain myelin content.
Interestingly, while a suspension of cells from the forebrain of these animals (Konat et
al., 1978) exhibited a 30 percent inhibition of total protein synthesis at 20 uM triethyl lead
chloride (the lowest concentration examined), a cell-free system prepared from the same tissue
was not affected by triethyl lead chloride concentrations as high as 200 uM. This result,
coupled with a similar, although not as severe, inhibitory effect of triethyl lead chloride on
oxygen consumption in the cell suspension (20 percent inhibition at 20 uM) would tend to indi-
cate that the inhibition of rat forebrain protein synthesis is related to an inhibition of
cellular energy-generating systems.
The effects of organolead compounds on various neurotransmitter systems have been inves-
tigated in adult mouse brain homogenates. Bondy et al. (1979a,b) demonstrated that micromolar
concentrations (5 uM) of tri-n-butyl lead (TBL) acetate were sufficient not only to cause a 50
percent decline in the high affinity uptake of GABA and DA in such homogenates, but also to
stimulate a 25 percent increase in GABA and DA release. These effects were apparently selec-
tive for DA neurons at lower concentrations, as only DA uptake or release was affected at 0.1
uM, albeit mildly so. The effect of TBL acetate on DA uptake appears to be specific, as there
is a clear dose-response relationship down to 1 uM TBL (Bondy and Agrawal, 1980) for inhibi-
tion (0-60 percent) of spiroperidol binding to rat striatal DA receptors. A concomitant
inhibition of adenyl cyclase in this dose range (50 percent) suggests that TBL may affect the
entire postsynaptic binding site for DA.
12.4.3.5 Accumulation and Retention of Lead in the Brain. All too infrequently, experimental
studies of the neurotoxic effects of lead exposure do not report the blood-lead levels
achieved by the exposure protocols used. Even less frequently reported are the concomitant
tissue levels found in brain or other tissues. From the recent information that is available,
however, it is possible to draw some limited conclusions about the relationship of exposure
concentrations to blood and brain lead concentrations. Table 12-9 calculates the blood lead/
brain lead ratios found in recent studies where such information was available. It can be
seen that, at exposure concentrations greater than 0.2 percent and for exposure periods longer
than birth until weaning (21 days in rats), the ratio generally falls below unity. This
suggests, that, even as blood lead levels reach a steady state and then fall due to excretion
or some other mechanism, lead continues to accumulate in brain.
12-150
-------
TABLE 12-9. INDEX OF BLOOD LEAD AND BRAIN LEAD LEVELS FOLLOWING EXPOSURE0
Species
(strain)
Suckling rat
(Charles
River-CD)
Suckling rat
(Charles
River)
Suckling rat
(Charles
River-CD)
Suckling rat
(Long- Evans)
Suckling rat
(Long-Evans)
Suckling rat
(Holtzman-
albino)
Suckling rat
Suckling rat
(Holtzman-
albino)
Suckling rat
(Long-Evans)
Exposure
0.0005% PbCl2
in water
PND 0-21
0.003% PbCl2
in water
PND 0-21
0.005% Pb(Ac)2
in water from
conception
0.01% Pb(Ac)2
in water from
conception
0.02% PbCl2
in water
PND 0-21
0.02% Pb(Ac)2
in water
PND 0-21
0.02% Pb(Ac)2
in water from
PND 0-21
0.05% Pb(Ac)2
in water
PND 0-21
0.1% Pb(Ac)2
in water
PND 0-21
0.2% Pb(Ac)2
in water
PND 0-21
0.2% Pb(Ac)2
in water
PND 0-21
0.2% Pb(Ac)2
in water
PND 0-21
Time of Blood lead,
assay ug/dl
PND 21
PND 21
PND 11
PND 30
PND 11
PND 30
PND 21
PND 10
PND 21
PND 21
PND 21
PND 21
PND 21
PND 21
PND 10
PND 21
12
21
22
18
35
48
36
21.7
25.2
29
12
20
65
47
49.6
89.4
Brain lead, Blood: brain
ug/100g lead ratio
8
11
3
11
7
22
25
6.3
13
29
20
50
65
80
19
82
1.5
1.9
7.0
1.6
5.0
2.2
1.4
3.4
1.9
1.0
0.6
0.4
1.0
0.6
2.6
1.1
Reference
Bull et al.
(1979)
Grant et al.
(1980)
Bull et al.
(1979)
Fox et al .
(1979)
Hastings
et al.
(1979)
Goldman
et al.
(1980)
Hastings et
al. (1979)
Goldman
et al.
(1980)
Fox et al .
(1979)
12-151
-------
TABLE 12-9. (continued)
Species
(strain)
Suckling rat
(Long-Evans)
Suckling rat
(Long-Evans)
Suckling mice
(ICR Swiss
albino)
Suckling rat
(Wistar)
Suckling rat
(Sprague-
Oawley)
Suckling rat
(Wistar albino)
Suckling rat
(Sprague-
Dawley)
Exposure
0.2% Pb(Ac)2
in water
PND 0-21
0.2% Pb(Ac)2
in water
PND 0-21
0.25% Pb(Ac)2
in water
PND 0-21
0.2% Pb(Ac)2
in water
PND 2-60
0.5% Pb(AC)2
in water
PND 2-60
0.25% Pb(Ac)2
in water from
gestation until
PND 42
0.5% Pb(Ac)2
in water
PND 0-21
1% Pb(Ac)2
in water
PND 0-21
0.5% Pb(Ac)2
in diet
PND 0-365
4% PbC03
in water
PND 0-27
Time of Blood lead,
assay ug/dl
PND 21
PND 21
PND 21
PND 30
PND 60
PND 30
PND 60
PND 42
PND 21
PND 21
PND 7
PND 21
PND 35
PND 49
PND 90
PND 180
PND 365
PND 27
65.0
65.1
72
115b
h
35D
308b
k
73D
87
70
91
70
335
291
94
76
78
103
—
Brain lead, Blood: brain
ug/lOOg lead ratio Reference
53
53
230
84
99
172
222
85
280
270
36
127
124
122
123
111
161
1.36
1.2
1.2
0.3
1.4
0.4
1.8
0.3
1.0
0.25
0.3
1.9
2.6
2.3
0.8
0.6
0.7
0.6
___
Fox et al.
(1977)
Cooper
et al.
(1980)
Modak et al.
(1978)
Shigeta
et al.
(1979)
Govoni
et al.
(1980)
MykkSnen et
al. (1979)
MykkSnen et
al. (1982)
Wince et al.
(1980)
12-152
-------
TABLE 12-9. (continued)
Species
(strain)
Suckling rat
(Sprague-
Dawley)
Suckling rat
(Long-Evans)
Young mice
(ICR Swiss
albino)
Weanling rats
(Long- Evans)
Adult rat
(Charles
River-CD)
Time of
Exposure assay
0.1 mg/kg Pb(Ac)2 PND 28
by gavage PND 42
PND 3-56 PND 56
25 mg/kg Pb(Ac)2 PND 15
by gavage
PND 2-14
75 mg/kg Pb(Ac)2 PND 15
by gavage
PND 2-14
0.25% Pb(Ac)2 PND 60
in water
PND 0-60
0.5% Pb(Ac)2 PND 60
in water
PND 0-60
1* Pb(Ac)2 PND 60
in water PND 0-60
0.0025% Pb(Ac)2
in water from
PND 22
0.005% Pb(Ac)2
in water from
PND 22
0.01% Pb(Ac)2
in water from
PND 22
0.05% Pb(Ac)2
in water from
PND 22
0.0005% Pb(Ac)2
in water for 21 days
Blood lead,
M9/dl
9.5
13.8
12.7
50
98
91
194
223
18
20
40
100
9
Brain lead,
Mg/ioog
12.1
11.1
10.2
40
60
410
360
810
7
30
50
120
10
Blood: brain
lead ratio
0.78
1.2
1.3
1.3
1.6
0.2
0.5
0.3
2.6
0.7
0.8
0.8
0.9
Reference
Collins et
al. (1984)
Jason and
Kellogg
(1981)
Modak et al.
(1978)
Cory-Slechta
et al.
(1985)
Bull et al.
(1979)
0.003% Pb(Ac)2
in water for 21 days
0.02% Pb(Ac)2
in water for 21 days
11
29
12
100
0.9
0.3
12-153
-------
TABLE 12-9. (continued)
Species
(strain)
Adult rat
(Wistar)
0.
in
Exposure
15% Pb(Ac)2
water for 3
Time of
assay
months
Blood lead,
Hg/dl
31
Brain lead,
ng/100g
12-18C
Blood:
lead
2.
brain
ratio
6-1.7c
Reference
Ewers and
Erbe
(1980)
0.4% Pb(Ac)2 69
in water for 3 months
1% Pb(Ac)2 122
in water for 3 months
16-34C 4.3-2.0c
37-72° 3.3-1.7c
Abbreviations:
PND: post-natal day
Pb(Ac)2: lead acetate
PbCl2: lead chloride
Expressed as ug Pb/lOOg blood.
cDepending on region.
Further evidence bearing on this was derived from a set of studies by Goldstein et al.
(1974), who reported that administration of a wide range of doses of radioactive lead nitrate
to one-month-old rats resulted in parallel linear increases in both blood and brain lead
levels during the ensuing 24 hours. This suggests that deposition of lead in brain occurs
without threshold and that, at least initially, it is proportional to blood lead concentra-
tion. However, further studies by Goldstein et al. (1974) followed changes in blood and brain
lead concentrations after cessation of lead exposure and found that, whereas blood lead levels
decreased dramatically (by an order of magnitude or more) during a 7-day period, brain lead
levels remained essentially constant over the one-week postexposure period. Thus, with even
intermittent exposures to lead, it is not unexpected that brain concentrations would tend to
remain the same or even to increase although blood lead levels may have returned to "normal"
levels. Evidence confirming this comes from findings of two studies: (1) Hammond (1971),
showing that EDTA administration causing marked lead excretion in urine of young rats did not
significantly lower brain lead levels in the same animals; and (2) Goldstein et al. (1974),
showing that although EDTA prevented the i_n vitro accumulation of lead into brain mito-
chondria, if lead was added first EDTA was ineffective in removing lead from the mitochondria.
These results, overall, indicate that, although lead may enter the brain in rough proportion
to circulating blood lead concentrations, it is then taken up by brain cells and tightly bound
into certain subcellular components (such as mitochondrial membranes) and retained there for
12-154
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quite long after Initial external exposure ceases and blood lead levels markedly decrease.
This may help to account for the persistence of neurotoxic effects of various types noted
above long after the cessation of external lead exposure.
The uptake of lead into specific neural and non-neuronal elements of the brain has also
been studied and provides insight into possible morphological correlates of certain lead
effects discussed above and below as being observed i_n vivo or i_n vitro. For example, Collins
et al. (1984) observed preferential accumulation of lead in the hippocampus of suckling rats
fed 0.1 mg/kg Pb(Ac)2 per day by gastric intubation from PND 3-56. In another study, Stumpf
et al. (1980), via autoradiographic localization of 210Pb, found that ependymal cells, glial
cells, and endothelial cells of brain capillaries concentrate and retain lead above background
levels for several days after injections of tracer amounts of the elements. These cells are
non-neural elements of brain important in the maintenance of "blood-brain barrier" functions,
and their uptake and retention of lead, even with tracer doses, provides evidence of a morpho-
logical basis by which lead effects on blood-brain barrier functions may be exerted. Again,
the retention of lead in these non-neuronal elements for at least several days after original
exposure points towards the plausibility of lead exerting effects on blood-brain barrier func-
tions long after external exposure ceases and blood lead levels decrease back toward normal
levels. Uptake and concentration of lead in the nuclei of some cortical neurons even several
days after administration of only a tracer dose of 210Pb was also observed by Stumpf et al.
(1980) and provide yet another plausible morphological basis by which neurotoxic effects might
be exerted by lead long after external exposure terminates and blood lead levels return to
apparently "normal" levels.
12.4.4 Integrative Summary of Human and Animal Studies of Neurotoxicity
An assessment of the impact of lead on human and animal neurobehavioral function raises a
number of issues. Among the key points addressed here are the following: (1) the internal
exposure levels, as indexed by blood lead levels, at which various adverse neurobehavioral
effects occur; (2) the reversibility of such deleterious effects; and (3) the populations that
appear to be most susceptible to neural damage. In addition, the question arises as to the
utility of using animal studies to draw parallels to the human condition.
12.4.4.1 Internal Exposure Levels at Which Adverse Neurobehavioral Effects Occur. Markedly
elevated blood lead levels are associated with neurotoxic effects (including severe, irrever-
sible brain damage as indexed by the occurrence of acute and/or chronic encephalopathic symp-
toms) in both humans and animals. For most adult humans, such damage typically does not occur
until blood lead levels exceed 120 ug/dl. Evidence does exist, however, for acute encephalo-
pathy and death occurring in some human adults at blood lead levels below 120 ug/dl, down to
12-155
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about 100 ug/dl. In children, effective blood lead levels for producing encephalopathy or
death are somewhat lower, encephalopathy signs and symptoms having been reported for some
children at blood lead levels as low as 80-100 ug/dl.
It should be emphasized that, once encephalopathy occurs, death is not an improbable out-
come, regardless of the quality of medical treatment available at the time of acute crisis.
In fact, certain diagnostic or treatment procedures themselves tend to exacerbate matters and
push the outcome toward fatality if the nature and severity of the problem are not fully rec-
ognized or properly diagnosed. It is also crucial to note the rapidity with which acute en-
cephalopathic symptoms can develop or death can occur in apparently asymptomatic individuals
or in those apparently only mildly affected by elevated body burdens of lead. It is not
unusual for rapid deterioration to occur, with convulsions or coma suddenly appearing and with
progression to death within 48 hours. This strongly suggests that, even in apparently asymp-
tomatic individuals, rather severe neural damage probably exists at high blood lead levels
although such damage is not yet overtly manifested in obvious encephalopathic symptoms. This
conclusion is further supported by numerous studies showing that children with high blood lead
levels (over 80-100 ug/dl), but not observed to manifest acute encephalopathic symptoms, are
permanently cognitively impaired, as are most children who survive acute episodes of frank
lead encephalopathy.
Growing evidence indicates that subencephalopathic lead intoxication in adults causes
various overt neurological signs and symptoms at blood lead levels as low (40-60 ug/dl) as
those at which other overt manifestations (e.g., gastrointestinal symptoms) of lead intoxica-
tion have been detected. In addition, among apparently asymptomatic, non-overtly lead-
intoxicated adults, often more subtle (but important) central and peripheral nervous system
effects, e.g. slowed nerve conduction velocities, have been observed at blood lead levels as
low as 30 ug/dl.
Other evidence confirms that various types of neural dysfunction exist in apparently
asymptomatic children across a broad range of blood lead levels. The body of studies on low-
or moderate-level lead effects on neurobehavioral functions, as summarized in Table 12-2, pre-
sents a rather impressive array of data pointing to that conclusion. At high exposure levels,
several studies point to average 5-point IQ decrements in asymptomatic children at average
blood levels of 50-70 ug/dl. Other evidence is indicative of average IQ decrements of up to
4 points being associated with blood levels in a 30-50 ug/dl range. Below 30 ug/dl, the evi-
dence for IQ decrements is mixed, with some studies showing no significant associations with
lead once other confounding factors are controlled. Still, the 1-2 point differences in IQ
generally seen with blood lead levels in the 15-30 ug/dl range are suggestive of small lead
effects that are typically dwarfed by other social factors. Moreover, the highly significant
12-156
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linear relationship between IQ and blood lead over the range of 6 to 47 ug/dl found in low-SES
Black children indicates that IQ effects may be detected without evident threshold even at
these low levels, at least in this population of children. In addition, other behavioral
(e.g., reaction time, psychomotor performance) and electrophysiological (altered EEG patterns,
evoked potential measures, and peripheral nerve conduction velocities) are consistent with a
dose-response function relating neurotoxic effects to lead exposure levels as low as 15-30
|jg/dl and possibly lower. Although the comparability of blood lead concentrations across
species is uncertain (see discussion below), animal studies show neurobehavioral effects in
rats and monkeys at maximal blood lead levels below 20 pg/dl; some studies demonstrate
residual effects long after lead exposure has terminated and blood lead levels have returned
to approximately normal levels.
Timing, type, and duration of exposure are important factors in both animal and human
studies. It is often uncertain whether observed blood lead levels represent the levels that
were responsible for observed behavioral deficits. Monitoring of lead exposures in pediatric
subjects in all cases has been highly intermittent or non-existent during the period of life
preceding neurobehavioral assessment. In most studies of children, only one or two blood lead
values are provided per subject. Tooth lead may be an important cumulative exposure index;
but its modest, highly variable correlation to blood lead, FEP, or external exposure levels
makes findings from various studies difficult to compare quantitatively. The complexity of
the many important covariates and their interaction with dependent measures of modest validi-
ty, e.g., IQ tests, may also account for many of the discrepancies among the different
studies.
The precise medical or health significance of the neuropsychological and electrophysio-
logical effects associated with low-level lead exposure as reported in the above studies is
difficult to state with confidence at this time. Observed IQ deficits and other behavioral
changes, although statistically significant in some studies, tend to be relatively small as
reported by the investigators, but nevertheless may still affect the intellectual development,
school performance, and social development of the affected children sufficiently to be regard-
ed as adverse. This would be especially true if such impaired intellectual development or
school performance and disrupted social development were reflective of persisting, long-term
effects of low-level lead exposure in early childhood. Although the issue of persistence of
such lead effects remains to be more clearly resolved, some study results reviewed above
suggest that significant low-level lead-induced neurobehavioral and electrophysiological
effects may, in fact, persist at least into later childhood. Animal studies also demonstrate
long-term neurobehavioral effects of relatively moderate- or low-level lead exposure, even
after blood lead concentrations have dropped to nearly normal levels.
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12.4.4.2 The Question of Irreversibility. Little research on humans is available on persis-
tence of effects. Some work suggests the possibility of reversing mild forms of peripheral
neuropathy in lead workers, but little is known regarding the reversibility of lead effects on
central nervous system function in humans. A series of studies on a group of lead-exposed
children indicate persistent relationships between blood lead and altered slow wave cortical
potentials at two- and five-year follow-ups. However, IQ deficits in the same group of sub-
jects were no longer evident at the five-year follow-up. Some work suggests that other mea-
sures of classroom performance may be more sensitive indicators of lead-induced effects in
older children. Prospective longitudinal studies on the developmental effects of lead are
needed to answer questions on the persistence or reversibility of neurotoxic effects of early
lead exposure.
Various animal studies provide evidence that alterations in neurobehavioral function may
be long-lived, with such alterations being evident long after blood lead levels have returned
to control levels. These persistent effects have been demonstrated in monkeys as well as rats
under a variety of learning performance test paradigms. Such results are also consistent with
morphological, electrophysiological, and biochemical studies on animals that suggest lasting
changes in synaptogenesis, dendritic development, myelin and fiber tract formation, ionic
mechanisms of neurotransmission, and energy metabolism.
12.4.4.3 Early Development and Susceptibility to Neural Damage. On the question of early
childhood vulnerability, the neurobehavioral data are consistent with morphological and bio-
chemical studies of the susceptibility of the heme biosynthetic pathway to perturbation by
lead. Various lines of evidence suggest that the order of susceptibility to neurotoxic effects
of lead is: young > adult, and female > male. Animal studies also have pointed to the peri-
natal period of ontogeny as a particularly critical time for a variety of reasons: (1) it is
a period of rapid development of the nervous system; (2) it is a period where good nutrition
is particularly critical; and (3) it is a period where the caregiver environment is vital to
normal development. However, the precise boundaries of a critical period for lead exposure are
not yet clear and may vary depending on the species and function or endpoint that is being
assessed. One analysis of lead-exposed children suggests that differing effects on cognitive
performance may be a function of the different ages at which children are subjected to neuro-
toxic exposures. Nevertheless, there is general agreement that human infants and toddlers
below the age of three years are at special risk because of jm utero exposure, increased
opportunity for exposure because of normal mouthing behavior of lead-containing objects, and
increased rates of lead absorption due to various factors, e.g., iron and calcium defici-
encies.
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12.4.4.4 Utility of Animal Studies in Drawing Parallels to the Human Condition. Animal mod-
els are used to shed light on questions where it would be impractical or ethically unaccept-
able to use human subjects. This is particularly true in the case of exposure to environmen-
tal toxins such as lead. In the case of lead, it has been most effective and convenient to
expose developing animals via their mothers' milk or by gastric gavage, at least until wean-
ing. Very often, the exposure is continued in the water or food for some time beyond weaning.
This approach does succeed in simulating at least two features commonly found in human expo-
sure: oral intake and exposure during early development. The preweaning postnatal period in
rats and mice is of particular relevance in terms of parallels with the first two years or so
of human brain development.
Studies using rodents and monkeys have provided a variety of evidence of neurobehavioral
alteration induced by lead exposure. In most cases these effects suggest impairment in
"learning," i.e., the process of appropriately modifying one's behavior in response to infor-
mation from the environment. Such behavior involves the ability to receive, process, and
remember information in various forms. Some studies indicate behavioral alterations of a more
basic type, such as delayed development of certain reflexes. Other evidence suggests changes
affecting rather complex behavior in the form of social interactions.
Most of the above effects are evident in rodents and monkeys with blood lead levels
exceeding 30 ug/dl, but some effects on learning ability are apparent even at maximum blood
lead exposure levels below 20 ug/dl. Can these findings with animals be generalized to
humans? Given differences between humans, rats, and monkeys in heme chemistry, metabolism,
and other aspects of physiology and anatomy, it is difficult to state what constitutes an
equivalent internal exposure level, much less an equivalent external exposure level (see
Hammond et al. (1985) for a discussion of this). For example, is a blood lead level of 30
ug/dl in a suckling rat equivalent to 30 ug/dl in a three-year-old child? Until an answer is
available for this question, i.e., until the function describing the relationship of exposure
indices in different species is available, the utility of animal models for deriving dose-
response functions relevant to humans will be limited.
Questions also exist regarding the comparability of neurobehavioral effects in animals
with human behavior and cognitive function. One difficulty in comparing behavioral endpoints
such as locomotor activity is the lack of a consistent operational definition. In addition to
the lack of standardized methodologies, behavior is notoriously difficult to "equate" or com-
pare meaningfully across species because behavioral analogies do not demonstrate behavioral
homologies. Thus, it is improper to assume, without knowing more about the responsible under-
lying neurological structures and processes, that a rat's performance on an operant condi-
tioning schedule or a monkey's performance on a stimulus discrimination task necessarily
corresponds directly to a child's performance on a cognitive function test. Nevertheless,
12-159
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interesting parallels in hyper-reactivity and increased response variability do exist between
different species, and deficits in performance by mammalian animals on various tasks are
probably indicative of altered CNS functions, which are likely to parallel some type of
altered CNS function in humans as well.
In terms of morphological findings, there are reports of hippocampal lesions in both
lead-exposed rats and humans that are consistent with a number of independent behavioral find-
ings suggesting an impaired ability to respond appropriately to altered contingencies for
rewards. That is, subjects with hippocampal damage tend to persist in certain patterns of
behavior even when changed conditions make the behavior inappropriate; the same sort of ten-
dency seems to be common to a number of lead-induced behavioral effects, including deficits in
passive avoidance, operant extinction, visual discrimination, and various other discrimination
reversal tasks. Other morphological findings in animals, such as demyelination and glial cell
decline, are comparable to human neuropathologic observations only at relatively high exposure
levels.
Another neurobehavioral endpoint of interest in comparing human and animal neurotoxicity
of lead is electrophysiological function. Alterations of electroencephalographic patterns and
cortical slow wave voltage have been reported for lead-exposed children, and various electro-
physiological alterations both iji vivo (e.g., in rat visual evoked response) and in vitro
(e.g., in frog miniature endplate potentials) have also been noted in laboratory animals.
Thus, far, however, these lines of work have not converged sufficiently to allow for much in
the way of definitive conclusions regarding electrophysiological aspects of lead neuro-
toxicity.
Biochemical approaches to the experimental study of lead effects on the nervous system
have been basically limited to laboratory animal subjects. Although their linkage to human
neurobehavioral function is at this point somewhat speculative, such studies do provide in-
sight on possible neurochemical intermediaries of lead neurotoxicity. No single neurotrans-
mitter system has been shown to be particularly sensitive to the effects of lead exposure;
lead-induced alterations have been demonstrated in various neurotransmitters, including
dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid. In addition, lead has been
shown to have subcellular effects in the central nervous system at the level of mitochondrial
function and protein synthesis. In particular, some work has indicated that delays seen in
cortical synaptogenesis and metabolic maturation following prenatal lead exposure may well
underlie the delayed development of exploratory and locomotor function seen in other studies of
the neurobehavioral effects of lead. Further studies on the correlation between human blood
lead values and lead-induced disruptions of tetrahydrobiopterin metabolism indicate that sub-
sequent interference with neurotransmitter formation may be linked to small reductions in
IQ scores.
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Given the difficulties in formulating a comparative basis for internal exposure levels
among different species, the primary value of many animal studies, particularly HI vitro
studies, may be in the information they can provide on basic mechanisms involved in lead
neurotoxicity. A number of key i_n vitro studies are summarized in Table 12-10. These stu-
dies show that significant, potentially deleterious effects on nervous system function occur
at i_n situ lead concentrations of 5 uM and possibly lower. This suggests that, at least in-
tracellularly or on a molecular level, there may exist essentially no threshold for certain
neurochemical effects of lead. The relationship between blood lead levels and lead concen-
trations at extra- or intracellular sites of action, however, remains to be determined.
Despite the problems in generalizing from animals to humans, both the animal and the
human studies show considerable internal consistency in that they both support a continuous
dose-response functional relationship between lead and neurotoxic biochemical, morphological,
electrophysiological, and behavioral effects.
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TABLE 12-10. SUMMARY OF KEY STUDIES OF IN VITRO LEAD EXPOSURE*
Preparation
Exposure
concentration
Results
Reference
ro
i
ro
Adult rat brain
Isolated microvessels
from rat brain
Adult mouse
brain homogenate
Adult rat striatum
Embryonic chick
brain cell culture
Brainstem and forebrain
slices from PND-22 rats
Adult rat
cerebellar homogenates
Adult rat
cerebellar mitochondria
Adult frog
nerve/muscle preparation
Isolated, perfused
bullfrog retina
0.1 MM Pb(Ac)2
0.1 uM Pb(Ac)2
0.1-5 pM TBL
1-5 MM TBL
3 MM (Et3Pb)Cl2
3 MM (Et3Pb)Cl2
3-5 MM Pb2*
5 MM Pb(Ac)2
5 MM Pb2+
5 MM Pb2+
35% inhibition of whole-
brain BH4 synthesis
Blockade of sugar-specific
transport sites in capil-
lary endothelial cells
1) 50% decline in high
affinity uptake of DA;
2) 25% increase in
release of DA
0-60% inhibition of spiro-
peridal binding to DA
receptors
50% reduction in no. of
cells exhibiting processes
Inhibition of leucine in-
corporation into myelin
proteins
Inhibition of adenylate
cyclase activity
Inhibition of respiration
Increase in frequency of
MEPP's (indicative of
depression of synaptic
transmission)
Depression of rod (but not
cone) receptor potentials
Purdy et al.
(1981)
Kolber et al.
(1980)
Bondy et al.
(1979a,b)
Bondy and Agrawal
(1980)
Grundt et al.
(1981)
Konat and Clausen
(1978, 1980);
Konat et al.
(1979)
Taylor et al.
(1978)
Gmerek et al.
(1981)
Kolton and Yaari
(1982)
Fox and Si 11man
(1979)
-------
TABLE 12-10. (continued)
Preparation
Exposure
concentration
Results
Reference
ro
Cerebellar slices
from PND-14 rats
In oculo culture of
cerebellar tissue
from PND-15 rats
Cell suspension from
forebrain of PND-22 rats
Adult rat cerebral 50
and cerebellar mitochondria
Adult rat caudate
synaptosomes
5-7 uM (Et3Pb)Cl2
5-10 uM Pb2+
20 uM (Et3Pb)Cl2
Pb(Ac)2
50 uM PbCl2
Capillary endothelial
cells from rat cere-
cortex
100 MM Pb(Ac)2
Inhibition of incorporation
of S04 and serine into
myelin galactolipids
"Disinhibition" of NE-
induced inhibition of
spontaneous activity in
Purkinje cells
30% inhibition of total
protein synthesis
Inhibition of respiration
8-fold+increase in binding
of Ca2 to mitochondria
(effectively increases
Ca2 gradient across ter-
minal membrane, thus in-
creasing synaptic trans-
mission without altering
firing rates)
Pb preferentially seques-
tered in mitochondria like
Ca2 (possible basis for
Pb-induced disruption of
transmembrane Ca2 transport)
Grundt and
Neskovic (1980)
Taylor et al.
(1978)
Konat et al.
(1978)
Holtzman et al.
(1978, 1980b)
Silbergeld and
Adler (1978)
Silbergeld et al.
(1980b)
*Abbreviations:
PND: postnatal day
Pb(Ac)2: lead acetate
PbCl2: lead chloride
Et3Pb: triethyl lead
TBL: tri-n-butyl lead
DA: dopamine
NE: norepinephrine
BH4: tetrahydrobiopterin
MEPP's: miniature endplate potentials
-------
12.5 EFFECTS OF LEAD ON THE KIDNEY
12.5.1 Historical Aspects
The first description of renal disease due to lead was published by Lancereaux (1862).
In a painter with lead encephalopathy and gout, Lancereaux noted tubulo-interstitial disease
of the kidneys at autopsy. Distinctions between glomerular and tubulo-interstitial forms of
kidney disease were not, however, clearly defined in the mid-nineteenth century. Ollivier
(1863) reported observations in 37 cases of lead poisoning with renal disease and thus intro-
duced the idea that lead nephropathy was a proteinuric disease, a confusion with primary
glomerular disease that persisted for over a century. Under the leadership of Jean Martin
Charcot, interstitial nephritis characterized by meager proteinuria in lead poisoning was
widely publicized (Charcot, 1874; Charcot and Gombault, 1881) but not always appreciated by
contemporary physicians (Danjoy, 1864; Geppert, 1882; Lorimer, 1886).
More than ninety years ago, the English toxicologist Oliver (1885, 1891) distinguished
acute effects of lead on the kidney from lead-induced chronic nephropathy. Acute renal
effects of lead were seen in persons dying of lead poisoning and were usually restricted to
non-specific changes in the renal proximal tubular lining cells. Oliver noted that a "true
interstitial nephritis" developed later, often with glomerular involvement.
In an extensive review of the earlier literature, Pejic (1928) emphasized that changes in
the proximal tubules, rather than the vascular changes often referred to in earlier studies
(Gull and Sutton, 1872), constitute the primary injury to the kidney in lead poisoning. Many
subsequent studies have shown pathological alterations in the renal tubule with onset during
the early or acute phase of lead intoxication. These include the formation of inclusion
bodies in nuclei of proximal tubular cells (Blackman, 1936) and the development of functional
defects as well as ultrastructural changes, particularly in renal tubular mitochondria.
Wedeen (1984) has extensively reviewed the history of lead poisoning and its relationship to
kidney disease.
12.5.2 Lead Nephropathy in Childhood
Dysfunction of the proximal tubule was first noted as glycosuria in the absence of hyper-
glycemia in childhood pica (McKhann and Vogt, 1926). Later it was shown that the proximal
tubule transport defect included aminoaciduria (Wilson et al., 1953). Subsequently, Chisolm
et al. (1955) found that the full Fanconi syndrome was present: glycosuria, aminoaciduria,
phosphaturia (with hypophosphatemia), and rickets. Proximal tubular transport defects ap-
peared only when blood lead levels exceeded 80 ug/dl. Generalized aminoaciduria was seen more
consistently in Chisolm's (1962, 1968) studies than were other manifestations of renal dys-
function. The condition was related to the severity of clinical toxicity, with the complete
Fanconi syndrome occurring in encephalopathic children when blood lead concentrations exceeded
12-164
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150 ug/dl (National Academy of Sciences, 1972). Children who were under three years of age
excreted 4-8.9 mg of lead chelate during the first day of therapy with CaNa2EDTA at 75 mg/kg
per day. The aminoaciduria disappeared after treatment with chelating agents and clinical
remission of other symptoms of lead toxicity (Chisolm, 1962). This is an important observa-
tion relative to the long-term or chronic effects of lead on the kidney.
In a group of children with slight lead-related neurological signs reported by Pueschel
et al. (1972), generalized aminoaciduria was found in 8 of 43 children with blood lead levels
of 40-120 ug/dl. Blood lead values in the children with aminoaciduria were not specifically
provided but presumably were among the highest found. It should be noted that the children
reported to have aminoaciduria in this study were selected because of a blood lead level of
£50 ug/dl or a provocative chelation test of >500 pg of lead chelate per 24 hours.
Although children are considered generally to be more susceptible than adults to the
toxic effects of lead, the relatively sparse literature on childhood lead nephropathy probably
reflects a greater clinical concern with the life-threatening neurologic symptoms of lead in-
toxication than with the transient Fanconi syndrome.
12.5.3 Lead Nephropathy in Adults
There are various lines of evidence in the literature that prolonged lead exposure in
humans can result in chronic lead nephropathy in adults. This evidence is reviewed below in
terms of six major categories: (1) lead nephropathy following childhood lead poisoning; (2)
"moonshine" lead nephropathy; (3) occupational lead nephropathy; (4) lead and gouty nephro-
pathy; (5) lead and hypertensive nephrosclerosis; and (6) general population studies.
Although a variety of methods have been used to assess body burdens of lead, the EDTA
lead-mobilization test has emerged as the most reliable index of cumulative lead stores (see
Chapter 10, Section 10.3.3). The reliability of this test is apparent under various condi-
tions. For example, Leckie and Tompsett (1958) showed that increasing the dosage of
CaNa2EDTA above 2 g/day intravenously had little effect on the amount of lead chelate excreted
by adults. They observed little difference in chelatable lead excretion when 1 g was compared
with 2 g intravenously. Similarly, the magnitude of lead chelated when 1 g is given intrave-
nously or 2 g intramuscularly (over 12 hr) appears to be the same (Albahary et al. , 1961;
Emmerson, 1963; Wedeen et al., 1975). Adult control subjects without undue lead absorption
excrete less than 650 |jg lead chelate during the first post-injection day if renal function is
normal, or over four days if renal function is severely reduced. Thus, in adults, urinary ex-
cretion of lead chelate in excess of about 600 ug over one or more days following 1-3 g
CaNa2EDTA administered intravenously or intramuscularly is considered indicative of excessive
past lead absorption. The level of reduction of glomerular filtration rate at which the EDTA
12-165
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lead-mobilization test is no longer reliable has not been precisely defined but probably ex-
ceeds a reduction of 85 percent (serum creatinine concentrations in excess of about 6 mg/dl).
12.5.3.1 Lead Nephropathy Following Childhood Lead Poisoning. Reports from Queensland,
Australia (Gibson et al., 1893; Nye, 1933; Henderson, 1954; Emmerson, 1963) point to a strong
association between severe childhood lead poisoning (including central nervous system
symptoms) and chronic nephritis in early adulthood. The Australian children sustained acute
lead poisoning because the houses around Brisbane were painted with white lead, which the
children ingested by direct contamination of their fingers or by drinking lead-sweetened rain
water as it flowed over the weathered surfaces. Two fingers brushed against the powdery paint
were shown to pick up about 2 mg of lead (Murray, 1939). Henderson (1954) followed up 401 un-
treated children who had been diagnosed as having lead poisoning in Brisbane between 1915 and
1935. Of these 401 subjects, death certificates revealed that 165 had died under the age of
40, 108 from nephritis or hypertension. This is greatly in excess of expected probabilities.
Information was obtained from 101 of the 187 survivors, and 17 of these had hypertension and/
or albuminuria.
The Australian investigators also established the validity of the EDTA lead-mobilization
test for the detection of excessive past lead absorption and further demonstrated that the
body lead stores were retained primarily in bone (Emmerson, 1963; Henderson, 1954; Inglis et
al., 1978). Bone lead concentrations averaged 94 ug/g wet weight in the young adults dying of
lead nephropathy in Australia (Henderson and Inglis, 1957; Inglis et al., 1978), compared with
mean values ranging from 14 to 23 ug/g wet weight in bones from non-exposed individuals
(Barry, 1975; Emmerson, 1963; Gross et al., 1975; Wedeen, 1982).
Attempts to confirm the relationship between childhood lead intoxication and chronic
nephropathy have not been successful in at least two studies in the United States. Most
children in the United States who suffer from overt lead toxicity do so early in childhood,
between the ages of 1 and 4, the source often being oral ingestion of flecks of wall paint and
plaster containing lead. Tepper (1963) found no evidence of increased chronic renal disease
in 139 persons with a well-documented history of childhood plumbism 20-35 years earlier at
the Boston Children's Hospital. The total study population comprised 165 patients (after re-
view of 524 case records) who met any two of the following criteria: 1) a definite history of
pica or use of lead nipple shields; 2) X-ray evidence of lead-induced skeletal alterations; or
3) characteristic symptoms of lead toxicity. No uniform objective measure of lead absorption
was reported. In 42 of the 139 subjects in question, clinical tests of renal function were
performed and included urinalysis, endogenous creatinine clearance, urine culture, urine con-
centrating ability, 24-hour protein excretion, and phenolsulfonphthalein excretion. Only one
patient was believed to have died of lead nephropathy; three with creatinine clearances under
90 ml/min were said to have had inadequate urine collections. Insufficient details concerning
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past lead absorption and patient selection were provided to permit generalized conclusions
from this report.
Chisolm et al. (1976) also found no evidence of renal disease in 55 adolescents known to
have been treated for lead intoxication 11-16 years earlier. This U.S. study was carried out
on adolescents between 12 and 22 years of age in the late 1960s. During acute toxicity in
early childhood, blood lead levels had ranged from 100 to 650 ug/dl; all had received immedi-
ate chelation therapy. Follow-up chelation tests performed with 1 g EDTA i.m. (with procaine)
approximately a decade later resulted in 24-hour lead-chelate excretion of less than 600 ug in
45 of 52 adolescents. Thus, an important distinction between the Australian group and those
patients in the United States studied by Chisolm et al. (1976) is that none of the latter sub-
jects showed evidence of increased residual body lead burden by the EDTA lead-mobilization
test. The absence of renal disease (as judged by routine urinalysis, blood serum urea nitro-
gen, serum uric acid, and creatinine clearance) led Chisolm et al. to suggest that lead toxi-
city in the Australian children may have been of a different type, with a more protracted
course than that experienced by the American children. On the other hand, chelation therapy
of the American children may have removed lead stored in bone and thus prevented the develop-
ment of renal failure later in life.
12.5.3.2 "Moonshine" Lead Nephropathy. In the United States, chronic lead nephropathy in
adults was first noted among illicit whiskey consumers in the southeastern states. The pre-
revolutionary tradition of homemade whiskey ("moonshine") was modernized during the Prohibi-
tion era for large-scale production. The copper condensers traditionally used in the illegal
stills were replaced by truck radiators with lead-soldered parts. Illegally produced whiskey
might contain up to 74 mg of lead per liter (Eskew et al., 1961). The enormous variability in
moonshine lead content has recently been reiterated in a study of 12 samples from Georgia, of
which five contained less than 10 ug/1 but one contained 5.3 mg/1 (Gerhardt et al., 1980).
Renal disease often accompanied by hypertension and gout was common among moonshiners
(Eskew et al., 1961; Morgan et al. , 1966; Ball and Sorensen, 1969). These patients usually
sought medical care because of symptomatic lead poisoning characterized by colic, neurological
disturbances, and anemia, although more subtle cases were sometimes detected by use of the
i.v. EDTA lead-mobilization test (Morgan, 1968; Morgan and Burch, 1972). While acute sympto-
matology, including azotemia, sometimes improved during chelation therapy, residual chronic
renal failure, gout, and hypertension frequently proved refractory, thus indicating underlying
chronic renal disease superimposed on acute renal failure due to lead (Morgan, 1975).
12.5.3.3 Occupational Lead Nephropathy. Although rarely recognized in the United States
(Brieger and Rieders, 1959; Anonymous, 1966; Greenfield and Gray, 1950; Johnstone, 1964;
Kazantzis, 1970; Lane, 1949; Malcolm, 1971; Mayers, 1947), occupational lead nephropathy,
often in association with gout and hypertension, was widely identified in Europe as a sequela
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to overt lead intoxication in the industrial setting (Albahary et al., 1961, 1965; Cramer et
al., 1974; Danilovic, 1958; Galle and Morel-Maroger, 1965; Lejeune et al., 1969; Li Us et al.,
1967, 1968; Radosevic et al., 1961; Radulescu et al. , 1957; Richet et al., 1964, 1966; Tara
and Francon, 1975; Vigdortchik, 1935). Some of the more important recent studies are summa-
rized here.
Richet et al. (1964) reported renal findings in eight lead workers, all of whom had re-
peated episodes of lead poisoning, including colic. Intravenous EDTA lead-mobilization tests
ranged from 587 to 5930 ug lead-chelate excretion per 24 hours. Four of these men had reduced
glomerular filtration rates, one had hypertension with gout, one had hypertension alone, and
one had gout alone. Proteinuria exceeded 200 mg/day in only one patient. Electron microscopy
showed intranuclear and cytoplasmic inclusions and ballooning of mitochondria in proximal
tubule cells. The presence of intranuclear inclusion bodies is helpful in establishing a re-
lationship between renal lesions and lead toxicity, but inclusion bodies are not always pre-
sent in persons with chronic lead nephropathy (Cramer et al., 1974; Wedeen et al., 1975,
1979).
Richet et al. (1966) subsequently recorded renal findings in 23 symptomatic lead workers
in whom blood lead levels ranged from 30 to 87 ug/dl. Six had diastolic pressures over 90
mm Hg, three had proteinuria exceeding 200 mg/day, and five had gout. In 5 of 21 renal biop-
sies, glomeruli showed minor hyalinization, but two cases showed major glomerular disease.
Interstitial fibrosis and arteriolar sclerosis were seen in all but two biopsies. Intra-
nuclear inclusion bodies were noted in 13 cases. Electron microscopy showed loss of brush
borders, iron-staining intracellular vacuoles, and ballooning of mitochondria in proximal
tubule epithelial cells.
Effective renal plasma flow, as measured by plasma clearance of para-aminohippurate
(C . ), was determined in 14 lead-poisoned Rumanian workers before and after chelation therapy
by Lilis et al. (1967). C . increased from a pre-treatment mean of 428 ml/min (significantly
less than the control mean of 580 ml/min) to a mean of 485 ml/min after chelation therapy (p
<0.02). However, no significant increase in glomerular filtration rate (as determined by en-
dogenous creatinine clearance) was found. Lilis et al. (1967) interpreted the change in
effective renal plasma flow as indicating reversal of the renal vasoconstriction that accom-
panied acute lead toxicity. Although neither blood lead concentrations nor long-term follow-
up observations of renal function were reported, it seems likely that most of these patients
suffered from acute, rather than chronic, lead nephropathy.
In a subsequent set of 102 cases of occupational lead poisoning studied by Lilis et al.
(1968), seven cases of clinically verified chronic nephropathy were found. In this group, en-
dogenous creatinine clearance was less than 80 ml/min two weeks or more after the last episode
of lead colic. The mean blood lead level approximated 80 ug/dl (range: 42-141 ug/dl.) All
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patients excreted more than 10 mg lead chelate over 5 days during therapy consisting of 2 g
CaNa2EDTA i.v. daily. Nephropathy was more common among those exposed to lead for more than
10 years than among those exposed for less than 10 years. Most of the Rumanian lead workers
had experienced lead colic, and 13 of 17 had persistent hypertension that followed the appear-
ance of renal failure by several years. Proteinuria was absent except in two individuals who
excreted 250 and 500 mg/1. Hyperuricemia was not evident in the absence of azotemia. In both
studies by Lilis et al. (1967, 1968), reduced urea clearance preceded reduced creatinine
clearance.
Cramer et al. (1974) examined renal biopsies from five lead workers exposed for 0.5-20
years in Sweden. Their blood lead levels ranged from 71 to 138 (jg/dl, with glomerular filtra-
tion rates ranging from 65 to 128 ml/min, but C . exceeding 600 ml/min in all. Although
plasma concentrations of valine, tyrosine, and phenylalanine were reduced, excretion of these
amino acids was not significantly different from controls. A proximal tubular reabsorptive
defect might, therefore, have been present without increased amino acid excretion because of
low circulating levels: increased fractional excretion may have occurred without increased
absolute amino acid excretion. Albuminuria and glycosuria were not present. Glomeruli were
normal by electron microscopy. Intranuclear inclusions in proximal tubules were found in two
patients with normal GFRs, and peri tubular fibrosis was present in the remaining three
patients who had had the longest occupational exposure (4-20 years).
Wedeen et al. (1975, 1979) reported on renal dysfunction in 140 occupationally exposed
men. These investigators used the EDTA lead-mobilization test (1 g CaNa2EDTA with 1 ml of
2 percent procaine given i.m. twice, 8-12 hr apart) to detect workers with excessive body lead
stores. In contrast to workers with concurrent lead exposure (Alessio et al., 1979), blood
lead measures have proven unsatisfactory for detection of past lead exposure (Baker et al. ,
1979; Havelda et al., 1980; Vitale et al. , 1975). Of the 140 workers tested, 113 excreted
1000 [jg or more of lead-chelate in 24 hr compared with a normal upper limit of 650 pg/day
(Albahary et al., 1961; Emmerson, 1973; Wedeen et al., 1975). Glomerular filtration rates
(GFR) measured by 125I-iothalamate clearance in 57 men with increased mobilizable lead re-
vealed reduced renal function in 21 (GFR less than 90 ml/min per 1.73 m2 body surface area).
When workers over age 55 or with gout, hypertension, or other possible causes of renal disease
were excluded, 15 remained who had previously unsuspected lead nephropathy. Their GFRs ranged
between 52 and 88 ml/min per 1.73 m2. Only three of the men with occult renal failure had
ever experienced symptoms attributable to lead poisoning. Of the 15 lead nephropathy
patients, one had a blood lead level over 80 M9/dl, three repeatedly had blood levels under 40
pg/dl, and eleven had blood levels between 40 and 80 (jg/dl at the time of the study. Thus,
blood lead levels were poorly correlated with degree of renal dysfunction. The failure of
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blood lead level to predict the presence of lead nephropathy probably stems from the indepen-
dence of blood lead from cumulative bone lead stores (Gross, 1981; Saenger et al., 1982a,b).
Percutaneous renal biopsies from 12 of the lead workers with reduced GFRs revealed focal
interstitial nephritis in six. Non-specific changes were present in proximal tubules, includ-
ing loss of brush borders, deformed mitochondria, and increased lysosomal bodies. Intra-
nuclear inclusion bodies were not found in the renal biopsies from these men who had experi-
enced long-term occupational exposure and who had had chelation tests shortly before biopsy.
In experimental animals, chelation results in the rapid disapperance of lead-induced intra-
nuclear inclusions (Goyer and Wilson, 1975). The detection of a variety of iimnunoglobulirt
deposits by fluorescent microscopy suggests (but does not prove) the possibility that some
stages of lead nephropathy in adults may be mediated by immune mechanisms.
Eight patients with pre-azotemic occupational lead nephropathy were treated with 1 g
CaNa2EDTA (with procaine) i.m. three times weekly for 6-50 months. In four patients, GFR rose
by 20 percent or more by the time the EDTA test had fallen to less than 850 ug Pb/day. The
rise in GFR was paralleled by increases in effective renal plasma flow (C .) during EDTA
treatment. These findings indicate that chronic lead nephropathy may be reversible by chela-
tion therapy, at least during the pre-azotemic phase of the disease (Wedeen et al., 1979).
However, much more information will have to be obtained on the value of long-term, low-dose
chelation therapy before this regimen can be widely recommended. There is, at present, no
evidence that interstitial nephritis itself is reversed by chelation therapy. It may well be
that only functional derangements are corrected and that the improvement in GFR is not accom-
panied by disappearance of tubulo-interstitial changes in kidney. Chronic volume depletion,
for example, might be caused by lead-induced depression of the renin-angiotension-aldosterone
system (McAllister et al., 1971) or by direct inhibition of (Na+, K+)ATPase-mediated sodium
transport (Nechay and Williams, 1977; Nechay and Saunders, 1978a,b,c; Raghavan et al., 1981;
Secchi et al., 1973). On the other hand, volume depletion would be expected to produce pre-
renal azotemia, but this was not evident in these patients. The value of chelation therapy in
chronic lead nephropathy once azotemia is established is unknown.
The prevalence of azotemia among lead workers has recently been confirmed in health sur-
veys conducted at industrial sites (Baker et al., 1979; Hammond et al., 1980; Landrigan et
al., 1982; Lilis et al., 1979, 1980). Interpretation of these data is, however, hampered by
the weak correlation generally found between blood lead levels and chronic lead nephropathy in
adults, the absence of matched prospective controls, and the lack of detailed diagnostic in-
formation on the workers found to have renal dysfunction. Moreover, blood serum urea nitrogen
(BUN) is a relatively poor indicator of renal function because it is sensitive to a variety of
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physiological variables other than GFR, including protein anabolism, catabolism, and hydra-
tion. Several other measures of renal function are more reliable than the BUN, including in
order of increasing clinical reliability: serum creatinine, endogenous creatinine clearance,
and 125I-iothalamate or inulin clearance. It should be noted that none of these measures of
GFR can be considered reliable in the presence of any acute illness such as lead colic or
encephalopathy. Elevated BUN in field surveys may, therefore, sometimes represent transient
acute functional changes rather than chronic intrinsic renal disease.
The variable susceptibility of the kidneys to the nephrotoxic effects of lead suggests
that environmental factors in addition to lead may participate in the expression of renal
damage. Industrial workers are often exposed to a variety of toxic materials, some of which,
such as cadmium (Buchet et al., 1980), are themselves nephrotoxic. In contrast to cadmium,
lead does not increase urinary excretion of beta-2-microglobulins (Batuman et al., 1981;
Buchet et al., 1980) or lysozyme (Wedeen et al., 1979) independently of increased low-molecu-
lar-weight proteinuria induced by renal failure itself. Multiple interactions between en-
vironmental toxins may enhance susceptibility to lead nephrotoxicity. Similarly, nephro-
toxicity may be modulated by reductions in 1,25-dihydroxyvitamin D3, increased 6-betahydroxy-
cortisol production (Saenger et al., 1981, 1982a,b), or immunologic alterations
(Gudbrandsson et al., 1981; Koller and Brauner, 1977; Kristensen, 1978; Kristensen and
Andersen, 1978). Reductions in dietary intake of calcium, copper, or iron similarly appear to
increase susceptibility to lead intoxication (Mahaffey and Michael son, 1980).
The slowly progressive chronic lead nephropathy resulting from years of relatively low-
dose lead absorption (i.e., insufficient to produce symptoms of acute intoxication) observed
in adults is strikingly different from the acute lead nephropathy arising from the relatively
brief but intense exposure arising from childhood pica. Typical acid-fast intranuclear inclu-
sions are, for example, far less common in the kidneys of adults (Cramer et al., 1974; Wedeen
et al., 1975). Although aminoaciduria has been found to be greater in groups of lead workers
than in controls (Clarkson and Kench, 1956; Goyer et al., 1972), proximal tubular dysfunction
is more difficult to demonstrate in adults with chronic lead nephropathy than in acutely ex-
posed children (Cramer et al., 1974). It should be remembered, however, that children with
the Fanconi syndrome have far more severe acute lead intoxication than is usual for workmen on
the job. In contrast to the reversible Fanconi syndrome associated with childhood lead poi-
soning, proximal tubular reabsorptive defects in occupationally exposed adults are uncommon
and subtle; clearance measurements are often required to discern impaired tubular reabsorption
in chronic lead nephropathy. Hyperuricemia is frequent among lead workers (Albahary et al.,
1965; Garrod, 1859; Hong et al., 1980; Landrigan et al., 1982), presumably a consequence of
specific lead inhibition of uric acid excretion, increased uric acid production (Emmerson et
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al., 1971; Granick et al., 1978; Ludwig, 1957), and pre-renal azotemia from volume depletion.
The hyperuricemia in adults contrasts with the reduced serum uric acid levels usually associa-
ted with the Fanconi syndrome in childhood lead poisoning. Although aminoaciduria and glyco-
suria are unusual in chronic lead nephropathy, Hong et al. (1980) reported a disproportionate
reduction in the maximum reabsorptive rate for glucose compared with para-aminohippurate (PAH)
in five of six lead workers they studied. PAH transport has not been consistently altered
beyond that expected in renal failure of any etiology (Hong et al., 1980; Wedeen et al.
1975). Biagini et al. (1977) have, however, reported a good negative linear correlation be-
tween the one-day EDTA lead-mobilization test and C . in 11 patients with histologic evidence
of lead-induced ultrastructural abnormalities in proximal tubules.
The differences between lead nephropathy in children and adults would not appear to be a
consequence of the route of exposure, since a case of pica in an adult (geophagic lead nephro-
pathy) studied by Wedeen et al. (1978) showed the characteristics of chronic rather than acute
lead nephropathy. Intranuclear inclusions were absent, and the GFR was reduced out of propor-
tion to the effective renal plasma flow.
12.5.3.4 Lead and Gouty Nephropathy. Renal disease in gout can often be attributed to well-
defined pathogenetic mechanisms including urinary tract stones and acute hyperuricemic nephro-
pathy with intratubular uric acid deposition (Bluestone et al., 1977). In the absence of
intra- or extra-renal urinary tract obstruction, the frequency, mechanism, and even the exist-
ence of a renal disease peculiar to gout remains in question. While some investigators have
described "specific" uric acid-induced histopathologic changes in both glomeruli and tubules
(Gonick et al., 1965; Sommers and Churg, 1982), rigorously defined controls with comparable
degrees of renal failure were not studied simultaneously. Specific histologic changes in the
kidneys in gout have not been found by others (Pardo et al. , 1968; Bluestone et al., 1977).
Glomerulonephritis, vaguely defined "pyelonephritis" (Heptinstall, 1974), or intra- and
extra-renal obstruction may have sometimes been confused with the gouty kidney, particularly
in earlier studies (Fineberg and Altschul, 1956; Gibson et al. , 1980b; Mayne, 1955; McQueen,
1951; Schnitker and Richter, 1936; Talbott and Terplan, 1960; Williamson, 1920).
The histopathology of interstitial nephritis in gout appears to be non-specific and can-
not usually be differentiated from that of pyelonephritis, nephrosclerosis, or lead nephro-
pathy on morphologic grounds alone (Barlow and Beilin, 1968; Bluestone et al., 1977; Greenbaum
et al. , 1961; Heptinstall, 1974; Inglis et al. , 1978). Indeed, renal histologic changes in
non-gouty hypertensive patients have been reported to be identical to those found in gout
patients (Cannon et al., 1966). In these hypertensive patients, serum uric acid levels paral-
leled the BUN.
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Confusion between glomerular and interstitial nephritis can in part be explained by the
tendency of proteinuria to increase as renal failure progresses, regardless of the underlying
etiology (Batuman et al., 1981). In the absence of overt lead intoxication it may, therefore,
be difficult to recognize surreptitious lead absorption as a factor contributing to renal
failure in gouty patients. Further, medullary urate deposits, formerly believed to be charac-
teristic of gout (Brown and Mallory, 1950; Mayne, 1955; McQueen, 1951; Fineberg and Altschul,
1956; Talbott and Terplan, I960), have more recently been reported in end-stage renal disease
patients with no history of gout (Cannon et al., 1966; Inglis et al., 1978; Linnane et al.,
1981; Ostberg, 1968; Verger et al., 1967). Whether such crystalline deposits contribute to,
or are a consequence of, renal damage cannot be determined with confidence. In the presence
of severe hyperuricemia (serum uric acid greater than 20 (jg/dl), intraluminal crystal deposi-
tion may produce acute renal failure because of tubular obstruction associated with grossly
visible medullary streaks (Emmerson, 1980). In chronic renal failure without gout or massive
hyperuricemia, the functional significance of such medullary deposits is unclear (Linnane et
al., 1981). Moreover, medullary microtophi, presumably developing around intraluminal depo-
sits, may extend into the renal interstitium, inducing foreign body reactions with giant cell
formation. Such deposits are sometimes overlooked in routine histologic preparations, as they
may be dissolved in aqueous fixatives. Their histologic identification requires alcohol fixa-
tion and deGalantha staining (Verger et al. , 1967). Because of the acid milieu, medullary
deposits are usually uric acid, while microtophi developing in the neutral pH of the renal
cortex are usually monosodium urate. Both amorphous and needle-like crystals have been demon-
strated in kidneys of non-gout and hyperuricemic patients, frequently in association with ar-
teriolonephrosclerosis (Inglis et al., 1978; Cannon et al., 1966; Ostberg, 1968). Urate depo-
sits, therefore, are not only not diagnostic, but may be the result, rather than the cause, of
interstitial nephritis. The problem of identifying unique characteristics of the gouty kidney
has been further confounded by the coexistence of pyelonephritis, diabetes mellitis, hyperten-
sion, and the aging process itself.
Although the outlook for gout patients with renal disease was formerly considered grim
(Talbott, 1949; Talbott and Terplan, 1960), more recent long-term follow-up studies suggest a
benign course in the absence of renovascular or other supervening disease (Fessel, 1979; Yu
and Berger, 1982; Yu, 1982). Over the past four decades the reported incidence of renal
disease in gout patients has varied from greater than 25 percent (Fineberg and Altschul, 1956;
Hench et al., 1941; Talbott, 1949; Talbott and Terplan, 1960; Wyngaarden, 1958) to less than 2
percent, as observed by Yu (1982) in 707 patients followed from 1970 to 1980. The low inci-
dence of renal disease in some hyperuricemic populations does not support the view that ele-
vated serum uric acid levels of the degree ordinarily encountered in gout patients are harmful
to the kidneys (Emmerson, 1980; Fessel, 1979; Ramsay, 1979; Reif et al. , 1981). Similarly,
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the failure of the xanthine oxidase inhibitor, allopurinol, to reverse the course of renal
failure in gout patients despite marked reductions in the serum uric acid (Bowie et al., 1967;
Levin and Abrahams, 1966; Ogryzlo et al., 1966; Rosenfeld, 1974; Wilson et al., 1967) suggests
that renal disease in gout may be due in part to factors other than uric acid. Some studies
have, however, suggested a possible slowing of the rate of progression of renal failure in
gout by allopurinol (Gibson et al., 1978, 1980a,b; Briney et al., 1975). While the contribu-
tion of uric acid to the renal disease of gout remains controversial, the hypothesized dele-
terious effect of hyperuricemia on the kidney has no bearing on other potential mechanisms of
renal damage in these patients.
Although hyperuricemia is universal in patients with renal failure, gout is rare in such
patients except when the renal failure is due to lead. Gout occurs in approximately half of
the patients with lead nephropathy (Emmerson, 1963, 1973; Ball and Sorensen, 1969; Richet et
al., 1965). Moreover, among gout patients in Scotland without known lead exposure, blood lead
levels were found to be higher than in non-gouty controls (Campbell et al. , 1978). The long
association of lead poisoning with gout raises the possibility that lead absorption insuffi-
cient to produce overt lead intoxication may, nevertheless, cause gout with slowly progressive
renal failure. Garrod (1859), Ball and Sorensen (1969), and Emmerson et al., (1971) demon-
strated that lead reduces uric acid excretion, thereby creating the internal milieu in which
gout can be expected. The mechanism of hyperuricemia in lead poisoning is, however, unclear.
Serum uric acid levels would be expected to rise in association with lead-induced pre-renal
azotemia; increased proximal tubule reabsorption of uric acid could result from reduced glo-
merular filtration rate due to chronic volume depletion. Increased tubular reabsorption of
uric acid in lead nephropathy has been suggested by the pyrazinamide suppression test
(Emmerson et al., 1971), but interpretation of this procedure was questioned (Holmes and
Kelley, 1974). Lead-induced inhibition of tubular secretion of uric acid, therefore, remains
another possible mechanism of reduced uric acid excretion. In addition, some investigators
have found increased uric acid excretion in saturnine gout patients, thereby raising the
possibility that lead increases uric acid production in addition to reducing uric acid
excretion (Emmerson et al., 1971; Ludwig, 1957; Granick et al., 1978).
To test the hypothesis that undetected lead absorption may sometimes contribute to
renal failure in gout, Batuman et al. (1981) administered the EDTA lead-mobilization test to
44 armed service veterans with gout and assessed their renal function. Individuals currently
exposed to lead (e.g., lead workers) were excluded. Collection of urine during the EDTA lead-
mobilization test was extended to three days because reduced GFR delays excretion of the lead
chelate (Emmerson, 1963). Note that the EDTA test does not appear to be nephrotoxic even for
patients with preexisting renal failure (Wedeen et al., 1983). Half of the gout patients had
normal renal function and half had renal failure as indicated by serum creatinines over 1.5
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mg/dl (mean = 3.0; standard error =0.4 mg/dl), reflecting approximately 70 percent reduction
in renal function. The groups were comparable with regard to age, duration of gout, incidence
of hypertension, and history of past lead exposure. The mean (and standard error) blood lead
concentration was 26 (± 3) jjg/dl in the patients with reduced renal function and 24 (± 3)
ug/dl in the gout patients with normal kidney function. The gout patients with renal dysfunc-
tion, however, excreted significantly more lead chelate than did those without renal dysfunc-
tion (806 ± 90 versus 470 ± 52 ug lead over 3 days). Ten control patients with comparable
renal failure excreted 424 ± 72 jjg lead during the 3-day EDTA test (2 g i.m.). The non-gout
control patients with renal failure had normal lead stores (Emmerson, 1973; Wedeen et al.,
1975), indicating that the excessive mobilizable lead in the gout patients with renal failure
was not a consequence of reduced renal function per se. The source of lead exposure in these
armed service veterans could not be determined with confidence. A history of transient occu-
pational exposure and occasional moonshine consumption was common among all the veterans, but
the medical histories did not correlate with either the EDTA lead-mobilization test or the
presence of renal failure. The relative contributions of airborne lead, industrial sources,
and illicit whiskey to the excessive body lead stores demonstrated by the EDTA lead-mobiliza-
tion test could not, therefore, be determined.
These studies suggest that excessive lead absorption may sometimes be responsible for the
gouty kidney in contemporary patients, as appeared to be the case in the past (Wedeen, 1981).
Although the EDTA lead-mobilization test cannot prove the absence of other forms of renal
disease, a positive EDTA test can indicate that lead may be a contributing cause of renal
failure when other known causes are excluded by appropriate diagnostic studies.
12.5.3.5 Lead and Hypertensive Nephrosclerosis. Hypertension '* another putative complica-
tion of excessive lead absorption that has a long and controversial history. In the older
literature hypertension was often linked to lead poisoning, frequently in association with
renal failure (Beevers et al. , 1980; Dingwall-Fordyce and Lane, 1963; Emmerson, 1963; Legge,
1901; Lorimer, 1886; Morgan, 1976; Oliver, 1891; Richet et al., 1966; Vigdortchik, 1935); but
a number of other investigators also failed to find such an association (Belknap, 1936;
Brieger and Rieders, 1959; Cramer and Dahlberg, 1966; Fouts and Page, 1942; Malcolm, 1971;
Mayers, 1947; Ramirez-Cervantes et al., 1978). Much more consistent evidence for associations
between lead exposure and hypertension has emerged, however, from numerous recent studies (as
discussed in the 1986 Addendum to this document). This includes epidemiological evidence
which suggests that hypertension is possibly mediated by lead-induced renal effects. Some of
the evidence pointing toward renal involvement is concisely reviewed below.
Among non-occupationally exposed individuals in Scotland, hypertension and serum uric
acid levels have been found to correlate with blood lead levels (Beevers et al., 1976). The
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kidneys of patients with chronic lead nephropathy may show uric acid deposits and the vas-
cular changes of "benign essential hypertension" even in the absence of gout and hypertension
(Cramer et al., 1974; Inglis et al., 1978; Morgan, 1976; Wedeen et al., 1975). In a long-term
follow-up study of 624 patients with gout, Yii and Berger (1982) reported that while hyper-
uricemia alone had no deleterious effect on renal function, decreased renal function was more
likely to occur in gout patients with hypertension and/or ischemic heart disease than in those
with uncomplicated gout.
Hypertension by itself is widely accepted as a cause of renal failure, although the renal
sequelae of moderate hypertension appear to be less dramatic than in the past (Kincaid-Smith
1982). In order to determine if unsuspected excessive body lead stores might contribute to
the renal disease of hypertension, 3-day EDTA (2 g i.m.) lead-mobilization tests were perform-
ed in hypertensive armed service veterans with and without renal failure (Batuman et al.
1983). A significant increase in mobilizable lead was found in hypertensive subjects with
renal disease compared to those without renal disease. Control patients with renal failure
again demonstrated normal mobilizable lead, thereby supporting the view that renal fail-
ure is not responsible for the excess mobilizable lead in patients with hypertension and renal
failure. These findings suggest that patients who would otherwise be deemed to have essential
hypertension with nephrosclerosis can be shown to have underlying lead nephropathy by the EDTA
lead-mobilization test when other renal causes of hypertension are excluded.
The mechanism whereby lead induces hypertension remains unclear. Although renal disease
particularly at the end-stage, is a recognized cause of hypertension, renal arteriolar histo-
logic changes may precede both hypertension and renal disease (Wedeen et al., 1975). Lead may
therefore induce hypertension by direct or indirect effects on the vascular system (see Sec-
tion 12.9.1 and the Addendum to this document).
Studies of hypertension in moonshine consumers have indicated the presence of hyporenin-
emic hypoaldosteronism. A blunted plasma renin response to salt depletion has been described
in lead poisoned patients; this response can be restored to normal by chelation therapy
(McAllister et al., 1971; Gonzalez et al., 1978; Sandstead et al., 1970a). The diminished
renin-aldosterone responsiveness found in moonshine drinkers could not, however, be demon-
strated in occupationally exposed men with acute lead intoxication (Campbell et al., 1979).
Although the impairment of the renin-aldosterone system appears to be independent of renal
failure and hypertension, hyporeninemic hypoaldosteronism due to lead might contribute to the
hyperkalemia (Morgan, 1976) and the exaggerated natriuresis (Fleischer et al., 1980) of some
patients with "benign essential hypertension." Since urinary kallikrein excretion is reduced
in lead workers with hypertension, it has been suggested that the decrease in this vasodilator
12-176
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may contribute to lead-induced hypertension (Boscolo et al., 1981). The specificity of kalli-
krein suppression in the renal and hypertensive manifestions of excessive lead absorption can-
not, however, be determined from available data, because lead workers without hyper-
tension and essential hypertensive patients without undue lead absorption also have reduced
urinary kallikrein excretion.
12.5.3.6 General Population Studies. Few studies have been performed to evaluate the possi-
ble harmful effects of lead on the kidneys in populations without suspected excessive lead ab-
sorption from occupational or moonshine exposure.
An epidemiological survey in Scotland of households with water lead concentrations in ex-
cess of WHO recommendations (100 M9/1) revealed a close correlation between water lead content
and blood lead and serum urea concentrations (Campbell et al., 1977). In 970 households lead
concentrations in drinking water ranged from <0.1 to >8.0 mg/1. After clinical and biochemi-
cal screening of 283 subjects from 136 of the households with water lead concentrations in ex-
cess of 100 ug/1, a subsample of 57 persons with normal blood pressure and elevated serum urea
(40 ug/dl) was compared with a control group of 54 persons drawn from the study group with
normal blood pressure and normal serum urea. The frequency of renal dysfunction in indivi-
duals with elevated blood lead concentratons (>41 ug/dl) was significantly greater than that
of age- and sex-matched controls.
Since 62 general practitioners took part in the screening, the subsamples may have come
from many different areas; however, it was not indicated if matching was done for place of re-
sidence. The authors found a significantly larger number of high blood lead concentrations
among the persons with elevated serum urea and claimed that elevated water lead concentration
was associated with renal insufficiency as reflected by raised serum urea concentrations.
This conclusion is difficult to accept since serum urea is not the method of choice for eva-
luating renal function. Despite reservations concerning use of the BUN for assessing renal
function (due to transient fluctuations), these findings are consistent with the view that ex-
cessive lead absorption from household water causes renal dysfunction. However, the authors
used unusual statistical methods and could not exclude the reverse causal relationship, i.e.,
that renal failure had caused elevated blood lead levels in their study group. A carefully
matched control population of azotemic individuals from low lead households would have been
helpful for this purpose. A more convincing finding in another subsample was a strong asso-
ciation between hyperuricemia and blood lead level. This was also interpreted as a sign of
renal insufficiency, but it may have represented a direct effect of lead on uric acid produc-
tion or renal excretion.
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Campbell et al. (1977) also found a statistically significant correlation between blood
lead concentration and hypertension. Tap-water lead did not, however, correlate with blood
lead among the hypertensive group, thus suggesting that other environmental sources of lead
may account for the presence of high blood lead concentrations among hypertensive persons in
Scotland (Beevers et al., 1976, 1980).
12.5.4 Mortality Data
Cooper and Gaffey (1975) analyzed mortality data available from 1267 death certificates
for 7032 lead workers who had been hired by 16 smelting or battery plants between 1900 and
1969. Standardized mortality ratios revealed an excess of observed over predicted deaths from
"other hypertensive disease" and "chronic nephritis and other renal sclerosis." The authors
concluded that "high levels of lead absorption such as occurred in many of the workers in this
series, can be associated with chronic renal disease." In an extension of this mortality
study covering the period 1971-1975 (Cooper, 1981), the excess of deaths from "other hyper-
tensive disease" and "chronic nephritis" was no longer evident. In the follow-up study,
deaths from major cardiovascular and renal diseases were "slightly higher than expected," but
did not reach statistical significance (Cooper, 1981). Cooper (1984) recently reexamined data
for a more rigorously selected subset of the same population (6819 workers versus 7032 origi-
nally) for the period 1947-1980. He found significantly greater than expected mortality for
both battery plant and lead smelter workers. These excess deaths appeared to result primarily
from malignant neoplasms (but not renal malignancies), chronic renal disease, and "ill-
defined" causes. Chronic renal disease reflected two general classifications: "other hyper-
tensive disease" and "chronic and unspecified nephritis." Most of these deaths occurred prior
to 1971, which accounts for the lack of such findings in Cooper's (1981) analysis of 1971-1975
data. Cooper (1984) noted that, although battery plant workers showed the greatest excess of
deaths from these causes, they did not show significantly elevated standardized mortality
ratios for hypertensive heart disease or stroke. Despite the lack of excess of renal carci-
noma in Cooper's analyses, kidney tumors have been found in lead-poisoned experimental animals
(see Section 12.7) and in at least two cases of occupationally exposed workers (Baker et al.,
1980; Lilis, 1981). Selevan et al. (1984) have also noted an increased, but not statistically
significant, incidence of renal cancer in a group of lead smelter workers.
In a more limited study of 241 Australian smelter employees who were diagnosed as lead
poisoned between 1928 and 1959 by a government medical board, 140 deaths were identified be-
tween 1930 and 1977 (McMichael and Johnson, 1982). Standard proportional mortality rates of
the lead-exposed workers compared with 695 non-lead-exposed employees revealed an overall
threefold excess in deaths due to chronic nephritis and a twofold excess in deaths due to
cerebral hemorrhage in the lead-exposed workers. Over the 47 years of this retrospective
12-178
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study, the number of deaths from chronic nephritis decreased from an initial level of 36 per-
cent to 4.6 percent among the lead-exposed workers, compared with a drop from 8.7 percent to
2.2 percent among controls. From 1965 to 1977 the age-standard!'zed mortality rates from
chronic nephritis were the same for the lead-worker and control groups, although both rates
were higher than the proportional mortality rate for the general population of Australian
males. The latter observation indicated that the excessive deaths from chronic nephritis
among lead-poisoned workers at the smelter had declined in recent decades.
Despite substantial evidence that lead produces interstitial nephritis in adults, the im-
pact of chronic lead nephropathy on the general population is unknown. The diagnosis of lead
nephropathy is rarely made in dialysis patients in the United States. The absence of the
diagnosis does not, however, provide evidence for the absence of the disease. Advanced renal
failure is usually encountered only many years after excessive lead exposure. Moreover, acute
intoxication may never have occurred, and neither heme enzyme abnormalities nor elevated blood
lead levels may be present at the time renal failure becomes apparent. The causal relation-
ship between lead absorption and renal disease may therefore not be evident. It is likely
that such cases of lead nephropathy have previously been included among other diagnostic
categories such as pyelonephritis, interstitial nephritis, gouty nephropathy, and hypertensive
nephrosclerosis. Increasing proteinuria as lead nephropathy progresses may also cause con-
fusion with primary glomerulonephritis. It should also be noted that the End Stage Renal
Disease Program (U.S. Health Care Financing Administration, 1982) does not even include the
diagnosis of lead nephropathy in its reporting statistics, regardless of whether the diag-
nosis is recognized by the attending nephrologist.
12.5.5 Experimental Animal Studies of the Pathophysiology of Lead Nephropathy
Laboratory studies of experimental animals have helped clarify a number of the mechanisms
underlying lead-induced nephropathy in humans. The following discussion will center on the
renal uptake and intracellular binding of lead, morphological alterations, various functional
changes, and biochemical effects associated with the renal toxicity of lead.
12.5.5.1 Lead Uptake by the Kidney. Lead uptake by the kidney has been studied in vivo and
in vitro using slices of renal tissue. Vander et al. (1977) performed renal clearance studies
in dogs two hours after a single i.v. dose of 0.1 or 0.5 mg lead acetate containing 1-3 mCi
of 203Pb or 1 hour after continous i.v. infusion of 0.1-0.15 mg/kg-hour. These investigators
reported that 43-44 percent of the plasma lead was ultrafiltrable, with kidney reabsorption
values of 89-94 percent for the ultrafiltrable fraction. A subsequent stop-flow analysis in-
vestigation by Victery et al. (1979a), using dogs given a single i.v. dose of lead acetate at
0.2 or 10.0 mg/kg, showed both proximal and distal tubular reabsorption sites for lead. Dis-
tal reabsorption was not linked to sodium chloride or calcium transport pathways. Proximal
12-179
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tubule reabsorption was demonstrated in all animals tested during citrate or bicarbonate
infusion. Another experiment by Victery et al. (1979b) examined the influence of acid-base
status on renal accumulation and excretion of lead in dogs given 0.5-50 ug/kg hour as an in-
fusion or in rats given access to drinking water containing 500 ppm lead for 2-3 months. They
showed that alkalosis increased lead entry into tubule cells via both luminal and basolateral
membranes, with a resultant increase in both renal tissue accumulation and urinary excretion
of lead. Similarly, acutely induced alkalosis increased lead excretion in rats previously
exposed through their drinking water. The authors concluded that earlier results from acute
exposure experiments on the renal handling of lead (Vander et al., 1977) were at least quali-
tatively similar to results of the chronic exposure experiments and that rats were an accept-
able model for investigating the effects of alkalosis on the excretion of lead following
chronic exposure.
Itn vitro studies (Vander et al., 1979) using slices of rabbit kidney incubated with 203Pb
acetate at lead concentrations of 0.1 or 1.0 pM over 180-minute time intervals showed that a
steady-state uptake of 203Pb by slices (ratio of slice to medium uptake in the range of 10-42)
was reached after 90 minutes and that lead could enter the slices as a free ion. Tissue slice
uptake was reduced by a number of metabolic inhibitors, thus suggesting a possible active
transport mechanism. Tin (Sn IV) markedly reduced 203Pb uptake into the slices but did not
affect lead efflux or para-aminohippurate accumulation. This finding raises the possibility
that Pb and Sn (IV) compete for a common carrier. Subsequent studies also using rabbit kidney
slices (Vander and Johnson, 1981) showed that co-transport of 203Pb into the slices in the
presence of organic anions such as cysteine, citrate, glutathione, histidine, or serum ultra-
filtrate was relatively small compared with uptake due to ionic lead.
In summary, it is clear from the above iji vivo and HI vitro studies on several different
animal species that renal accumulation of lead is an efficient process that occurs in both
proximal and distal portions of the nephron and at both luminal and basolateral membranes.
The transmembrane movement of lead appears to be mediated by an uptake process that is subject
to inhibition by several metabolic inhibitors and the acid-base status of the organism.
12.5.5.2 Intracellular Binding of Lead in the Kidney. The bioavailability of lead inside
renal tubule cells under low or 203Pb-tracer exposure conditions is mediated in part by bind-
ing to several high-affinity cytosolic binding proteins (Oskarsson et al. , 1982; Mistry et
al., 1982) and, at higher exposure conditions, by the formation of cytoplasmic and intra-
nuclear inclusion bodies (Goyer et al., 1970a). These inclusion bodies have been shown by
both cell fractionation (Goyer et al., 1970a) and X-ray microanalysis (Fowler et al., 1980) to
contain the highest intracellular concentrations of lead. Saturation analysis of the renal
12-180
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63,000 dalton (63K) cytosolic binding protein has shown that it possesses an approximate dis-
-8
sociation constant (K.) of 10 M (Mistry et al., 1982). These data quantify the high-affi-
nity nature of this protein for lead and explain the previously reported finding (Oskarsson et
al., 1982) that this protein constitutes a major intracellular lead-binding site in the kidney
cytosol. Biochemical studies on the protein components of isolated rat kidney intranuclear
inclusion bodies have shown that the main component has an approximate molecular weight of 27K
(Moore et al., 1973) or 32K (Shelton and Egle, 1982) and that it is rich in two dicarboxylic
amino acids, glutamate and aspartate (Moore et al. , 1973). The isoelectric point of the main
nuclear inclusion body protein has been reported to be pi =6.3 and appeared from two-dimen-
sional gel analysis to be unique to nuclei of lead-injected rats (Shelton and Egle, 1982).
The importance of the inclusion bodies resides with the suggestion (Goyer et al., 1970a; Moore
et al., 1973; Goyer and Rhyne, 1973) that, since these structures contain the highest intra-
cellular concentrations of lead in the kidney proximal tubule and hence account for much of
the total cellular lead burden, they sequester lead to some degree away from sensitive renal
organelles or metabolic (e.g., heme biosynthetic) pathways until their capacity is exceeded.
The same argument would apply to the high-affinity cytosolic lead-binding proteins at lead ex-
posure levels below those that cause formation of inclusion bodies. It is currently unclear
whether lead-binding to these proteins is an initial step in the formation of the cytoplasmic
or nuclear inclusion bodies (Oskarsson et al., 1982).
12.5.5.3 Pathological Features of Lead Nephropathy. The main morphological effects of lead
in the kidney are manifested in renal proximal tubule cells and interstitial spaces between
the tubules. A summary of morphological findings from some recent studies involving a number
of animal species is given in Table 12-11. In all but one of these studies, formation of in-
tranuclear inclusion bodies is a common pathognomic feature for all species examined. In ad-
dition, proximal tubule cell cytomegaly and swollen mitochondria with increased numbers of
lysosomes were also observed in two of the chronic exposure studies (Fowler et al., 1980; Spit
et al., 1981). Another feature reported in three of these studies (Mass et al., 1964; White,
1977; Fowler et al., 1980) was the primary localization of morphological changes in the
straight (S3) segments of the proximal tubule, thereby indicating that not all cell types of
the kidney are equally involved in the toxicity of lead to this organ. Interstitial fibrosis
has also been reported in rabbits (Hass et al., 1964) given diets containing 0.5 percent lead
acetate for up to 55 weeks and in rats (Goyer, 1971) given drinking water containing lead ace-
tate for 9 weeks.
12.5.5.4 Functional Studies
12.5.5.4.1 Renal blood flow and glomerular filtration rate. Studies by Aviv et al. (1980)
concerning the impact of lead on renal function as assessed by renal blood flow (RBF) and
glomerular filtration rate (GFR) have reported significant (p <0.01) reductions in both of
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TABLE 12-11. MORPHOLOGICAL FEATURES OF LEAD MEPHROPATHY IN VARIOUS SPECIES
CO
Species
Rabbit
Rat
Dog
Monkey
Rat
Rabbit
Ringed
dove
Nuclear
Pb dose regi*en inclusions
0.5X Pb acetate in +
diet for up to 55
weeks
IX Pb in drinking water +
for 9 weeks
50 ug/kg by gavage for +
5 weeks
0, 1.5, 6.0, 15 ugAiay *
in ad lib. drinking water
6 days/week for 9 months
0, 0.5, 5, 25, 50, 250 +
pp> in ad lib. drinking
water
0, 0.25, 0.50 MO/kg
by subcutaneous injection
3 days/wk for 14 weeks
100 ug/al +
in ad lib. drinking
water
Morphological findings*
1 ncreased
•itochondrial Increased
swelling Jysosomes
ND ND
+ ND
ND ND
NO ND
+
+
+
Interstitial
fibrosis Reference
+ Hass et al. (1964)
+ Goyer (1971)
ND White (1977)
ND Colle et al. (1980)
Fowler et al. (1980)
Spit et al. (1981)
Kendall et al. (1981)
*HD = Not determined; + = positive finding; - = negative finding.
-------
these parameters in rats at 3 and 16 weeks after termination of exposure to 1 percent lead
acetate in drinking water. A statistically significant (p <0.05) reduction of GFR has also
been recently described in dogs 2.5-4 hours after a single i.v. dose of lead at 3.0 mg/kg
(Victery et al., 1981). In contrast, studies by others (Johnson and Kleinman, 1979; Hammond
et al., 1982) were not able to demonstrate a reduction in GFR or RBF using the rat as a model.
The reasons behind these reported differences are currently unclear but may be related to
differences in experimental design, age of subjects, or other variables.
12.5.5.4.2 Tubular function. Exposure to lead has also been reported to produce tubular dys-
function (Studnitz and Haeger-Aronsen, 1962; Goyer, 1971; Mouw et al., 1978; Suketa et al.,
1979; Victery et al., 1981, 1982a,b, 1983). An early study (Studnitz and Haeger-Aronsen,
1962) reported aminoaciduria in rabbits given a single dose of lead at 125 mg/kg, with urine
collected over a 15-hour period. Goyer et al. (1970b) described aminoaciduria in rats follow-
ing exposure to 1 percent lead acetate in the diet for 10 weeks. Wapnir et al. (1979) con-
firmed a mild hyperaminoaciduria in rats injected with lead at 20 mg/kg five times a week for
six weeks but found no changes in urinary excretion of phosphate or glucose.
Other studies (Mouw et al., 1978; Suketa et al., 1979; Victery et al. , 1981, 1982a,b,
1983) have focused attention on increased urinary excretion of electrolytes. Mouw et al.
(1978) reported increased urinary excretion of sodium, potassium, calcium, and water in dogs
given a single intravenous injection of lead at 0.6 or 3.0 mg/kg over a 4-hour period. This
effect occurred despite a constant GFR, which indicates decreased tubular reabsorption of
these substances. Suketa et al. (1979) treated rats with a single oral dose of lead at 0, 5,
50, or 200 mg/kg and killed the animals at 0, 6, 12, or 24 hours after treatment. A dose-
related increase in urinary sodium, potassium, and water was observed over time. Victery et
al. (1981, 1982a,b, 1983) studied zinc excretion in dogs over a 4-hour period following an
intravenous injection of lead at 0.3 or 3.0 mg/kg. These investigators reported maximal in-
creases in zinc excretion of 140 ng/min at the 0.3 mg/kg dose and 300 ng/min at the 3.0 mg/kg
dose at the end of the 4-hour period. In contrast, studies by Mouw et al. (1978) showed no
changes in urinary excretion of sodium or potassium. Urinary protein and magnesium excretion
were also unchanged.
The results of the above studies indicate that acute or chronic lead treatment is capable
of producing tubular dysfunction in various mammalian species, as manifested by increased uri-
nary excretion of amino acid nitrogen, water, and some ions such as Zn2 , Ca2 , Na , and K .
12.5.6 Experimental Studies of the Biochemical Aspects of Lead Nephrotoxicity
12.5.6.1 Membrane Marker Enzymes and Transport Functions. The biochemical effects of lead in
the kidney appear to be preferentially localized in the cell membranes and mitochondrial and
nuclear compartments following either acute or chronic lead exposure regimens.
12-183
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Oral exposure of rats to lead acetate in the diet at concentrations of 1-2 percent for
10-40 weeks was found to produce no significant changes in the water content of renal slices
or in the accumulation of para-aminohippurate or tetraethyl-ammonium. However, tissue glucose
synthesis at 40 weeks and pyruvate metabolism were both significantly (p <0.05) reduced
(Hirsch, 1973).
Wapnir et al. (1979) examined biochemical effects in kidneys of rats injected with lead
acetate (20 mg/kg) five days per week for six weeks. They observed a significant (p <0.05)
reduction in renal alkaline phosphatase activity and an increase in (Mg2 )-ATPase, but no sig-
nificant changes in (Na ,K )-ATPase, glucose-6-phosphatase, fructose 1-6 diphosphatase, tryp-
tophan hydroxylase, or succinic dehydrogenase. These findings indicated that preferential
effects occurred only in marker enzymes localized in the brush border membrane and mitochon-
drial inner membrane. Suketa et al. (1979) reported marked (50-90 percent) decreases in renal
(Na ,K )-ATPase at 6-24 hours following a single oral administration of lead acetate at a dose
of 200 mg/kg. A later study (Suketa et al., 1981) using this regimen showed marked decreases
in renal (Na , K )-ATPase but no significant changes in (Mg2 )-ATPase after 24 hours, thus in-
dicating inhibition of a cell membrane marker enzyme prior to changes in a mitochondrial
marker enzyme.
12.5.6.2 Mitochondrial Respiration/Energy-Linked Transformation. Effects of lead on renal
mitochondrial structure and function have been studied by a number of investigators (Goyer,
1968; Goyer and Krall, 1969a,b; Fowler et al., 1980, 1981a,b). Examination of proximal tubule
cells of rats exposed to drinking water containing 0.5-1.0 percent lead acetate for 10 weeks
(Goyer, 1968; Goyer and Krall, 1969a,b) or 250 ppm lead acetate for 9 months (Fowler et al. ,
1980) has shown swollen proximal tubule cell mitochondria iji situ. Common biochemical find-
ings in these studies were decreases in respiratory control ratios (RCR) and inhibition of
state-3 respiration, which was most marked for NAD-linked substrates such as pyruvate/malate.
Goyer and Krall (1969a,b) found these respiratory effects to be associated with a decreased
capacity of mitochondria to undergo energy-linked structural transformation.
_4
It) vitro studies (Garcia-Canero et al., 1981) using 10 M lead demonstrated decreased
renal mitochondrial membrane transport of pyruvate or glutamate associated with decreased res-
piration for these two substrates. Other ui vitro studies (Fowler et al., 1981a,b) have shown
decreased renal mitochondrial membrane energization as measured by the fluorescent probes
l-anilino,-8 napthalenesulfonic acid (ANS) or ethidium bromide following exposure to lead
_5 _3
acetate at concentrations of 10 -10 M lead. High amplitude mitochondrial swelling was also
observed by light scattering.
The results of the above studies indicate that lead produces mitochondrial swelling both
irj situ and jm vitro, associated with a decrease in respiratory function that is most marked
12-184
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for RCR and state-3 respiration values. The structural and respiratory changes appear to be
linked to lead-induced alteration of mitochondrial membrane energization.
12.5.6.3 Renal Heme Biosynthesis. There are several reports concerning the effects of lead
on renal heme biosynthesis following acute or chronic exposure (see Table 12-12). Silbergeld
et al. (1982) injected rats with lead at 10 umol/kg per day for three days and examined ef-
fects on several tissues including kidney. These investigators found an increase in
6-aminolevulinic acid synthetase (ALA-S) following acute injection and no change following
chronic exposure (first indirectly via their dams' drinking water containing lead at 10 mg/ml
until 30 days of age and then directly via this drinking water until 40-60 days of age).
Renal tissue content of 6-aminolevulinic acid (ALA) was increased in both acutely and chroni-
cally exposed rats. Renal 6-aminolevulinic acid dehydrase (ALA-D) was found to be inhibited
in both acute and chronic treatment groups. Gibson and Goldberg (1970) injected rabbits s.c.
with lead acetate at doses of 0, 10, 30, 150, or 200 mg/week for up to 24 weeks. The mito-
chondrial enzyme ALA-S in kidney was found to show no measurable differences from control
levels. Renal ALA-D, which is found in the cytosol fraction, showed no differences from con-
trol levels when glutathione was present but was significantly reduced (p <0.05) to 50 percent
of control values for the pooled lead-treated groups when glutathione was absent. Mitochon-
drial heme synthetase (ferrochelatase) was not significantly decreased in lead-treated versus
control rabbits, but this enzyme in the kidney was inhibited by 72 and 94 percent at lead-
_4 -3
acetate concentrations of 10 and 10 M lead, respectively. Accumulation (12-15 fold) of
both ALA and porphobilinogen (PBG) was also observed in kidney tissue of lead-treated rabbits
relative to controls. Zawirska and Medras (1972) injected rats with lead acetate at a dose of
3 ing/day for up to 60 days and noted a similar renal tissue accumulation of uroporphyrin,
coproporphyrin, and protoporphyrin. A study by Fowler et al. (1980) using rats exposed
through 9 months of age to 50 or 250 ppm lead acetate in drinking water showed significant
inhibition of the mitochondrial enzymes ALA-S and ferrochelatase but no change in the activity
of the cytosolic enzyme ALA-D. Similar findings have been reported for ALA-D following acute
i.p. injection of lead acetate at doses of 5-100 mg/kg at 16 hours prior to sacrifice (Woods
and Fowler, 1982). In the latter two studies, reduced glutathione was present in the assay
mixture.
To summarize the above studies, the pattern of alteration of renal heme biosynthesis by
lead is somewhat different from that usually observed with this agent in other tissues (see
Section 12.3). In general, renal ALA-D does not seem to be inhibited much by lead except
under conditions of high-level exposure (Table 12-12). Such a finding could result from the
presence of the recently described high affinity cytosolic lead-binding proteins (Oskarsson et
al., 1982; Mistry et al., 1982) in the kidney and/or the formation of lead-containing
intranuclear inclusion bodies in this tissue (Goyer, 1971; Fowler et al., 1980), which would
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TABLE 12-12. EFFECTS OF LEAD EXPOSURE ON ASPECTS OF RENAL HEME BIOSYNTHESIS
Parameter Affected8
Species
Rabbit
Rat
Rat
Rat
(dams)
(newborns)
Rat
(dams)
(sucklings)
Rat
Rat
Rat
Pb dose regimen ALA-S ALA-D
10, 30, 150, 200 NC ±*
mg/kg per wk (s.c.)
3 mg/day NO NO
(s.c.)
10, 100, 1000, NO *
5000 ppm in
d.w. for 3 wks
10 ppm in d.w. ND NC
during:
3 wks before mating
3 wks of pregnancy
3 wks after delivery
ND NC
100 ppm in d.w. ND NC
for 3 wks
ND NC
0.5, 5, 25, 50, 250 * NC
ppm in d.w. for
9 months
5, 25, 50, 100 mg/kg ND NC
(i.p.) 16 hrs prior
to sacrifice
10 umol/kg/(i.p.) t 4-
for 3 days
10 mg/ml in d.w. NC *
for 10-30 days
Renal tissue
FC porphyrins
NC t ALA, PBG
(12-lSx)
ND t uro-,
copro- , proto-
porphyrins
ND t at 1000 and
5000 ppm;
t ALA-urine
ND NC
ND t
ND NC
ND t
4- ND
ND ND
ND t ALA
ND t ALA
Reference
Gibson and Gold-
berg (1970)
Zawirska and
Medras (1972)
Buchet et al.
(1976)
Hubermont
et al. (1976)
Reels et al.
(1977)
Fowler et al
(1980)
Woods and
Fowler (1982)
Silbergeld
et al. (1982)
t = increased; 4. = decreased; ± = effect depends on
relative to controls; ND = not determined.
s.c. - subcutaneous; i.p. - intraperitoneal; d.w. -
CFC - ferrochelatase.
conditions of assay; NC = no change
drinking water.
12-186
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sequester most of the intracellular lead away from other organelle compartments until the
capacity of these mechanisms is exceeded. Based on the observations of Gibson and Goldberg
(1970), tissue or assay concentrations of glutathione may also be of importance to the effects
of lead on this enzyme. The observed lack of ALA-S induction in kidney mitochondria reported
in the above studies may have been caused by decreased mitochondrial protein synthesis
capacity or, as previously suggested (Fowler et al., 1980), by overwhelming inhibition of this
enzyme by lead, such that any inductive effects were not measurable. Further research is
needed to resolve these questions.
12.5.6.4 Alteration of Renal Nucleic Acid/Protein Synthesis. A number of studies have shown
marked increases in renal nucleic acid or protein synthesis following acute or chronic expo-
sure to lead acetate. One study (Choie and Richter, 1972a) conducted on rats given a single
intraperitoneal injection of lead acetate showed an increase in 3H-thymidine incorporation. A
subsequent study (Choie and Richter, 1972b) involved rats given intraperitoneal injections of
1-7 mg lead once per week over a 6-month period. Autoradiography of 3H-thymidine incorpora-
tion into tubule cell nuclei showed a 15-fold increase in proliferative activity in the lead-
treated rats relative to controls. The proliferative response involved cells both with and
without intranuclear inclusions. Follow-up autoradiographic studies in rats given three
intraperitoneal injections of lead acetate (0.05 mg/kg) 48 hours apart showed a 40-fold
increase in 3H-thymidine incorporation 20 hours after the first lead dose and 6 hours after
the second and third doses.
Choie and Richter (1974a) also studied mice given a single intracardiac injection of lead
(5 ug/g) and demonstrated a 45-fold maximal increase in DNA synthesis in proximal tubule
cells as judged by 3H-thymidine autoradiography 33 hours later. This increase in DNA synthe-
sis was preceded by a general increase in both RNA and protein synthesis (Choie and Richter,
1974b). The above findings were essentially confirmed with respect to lead-induced increases
in nucleic acid synthesis by Cihak and Seifertova (1976), who found a 13-fold increase in
3H-thymidine incorporation into kidney nuclei of mice 4 hours after an intracardiac injection
(5 pg/g) of lead acetate. This finding was associated with a 34-fold increase in the
mitotic index but no change in the activities of thymidine kinase or thymide monophosphate
kinase. Stevenson et al. (1977) have also reported a 2-fold increase in 3H-thymidine or
14C-orotic acid incorporation into kidney DNA or RNA of rats given a single intraperitoneal
injection of lead chloride three days earlier.
The above studies clearly demonstrate that acute or chronic administration of lead by
injection stimulates renal nucleic acid and protein synthesis in kidneys of rats and mice.
The relationship between this proliferative response and formation of intranuclear inclusion
bodies is currently unknown; nor is the basic mechanism underlying this response and the
formation of renal adenomas in rats and mice following chronic lead exposure understood.
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12.5.6.5 Lead Effects on the RenirrAngiotension System. A study by Mouw et al. (1978) used
dogs given a single intravenous injection of lead acetate at doses of 0.6 or 3.0 mg/kg and
observed over a 4-hour period. Subjects showed a small but significant decrease in plasma
renin activity (PRA) at 1 hour, followed by a large and significant (p <0.05) increase from
2.5 to 4.0 hours. Follow-up work (Goldman et al., 1981) using dogs given a single intravenous
injection of lead acetate at 3.0 mg/kg showed changes in the renin-angiotensin system over
a 3-hour period. The data demonstrated an increase in PRA, but increased renin secretion oc-
curred in only three of nine animals. Hepatic extraction of renin was virtually eliminated in
all animals, thus providing an explanation for the increased blood levels of renin. Despite
the large observed increases in PRA, blood levels of angiotensin II (All) did not increase
after lead treatment. This suggests that lead inhibited the All converting enzyme.
Exposure of rats to drinking water containing 0.5 mg Pb/tnl for three weeks to five months
(Fleischer et al., 1980) produced an elevation of PRA after six weeks of exposure in those
rats on a sodium-free diet. No change in plasma renin substrate (PRS) was observed. At five
months, PRA was significantly higher in the lead-treated group on a 1-percent sodium chloride
diet, but the previous difference in renin levels between animals on an extremely low-sodium
(1 meq) versus 1-percent sodium diet had disappeared. The lead-treated animals had a reduced
ability to decrease sodium excretion following removal of sodium from the diet.
Victery et al. (1982a) exposed rats to lead jri utero and to drinking water solution con-
taining 0, 100, or 500 ppm lead as lead acetate for six months. Male rats on the 100 ppm lead
dose became significantly hypertensive at 3.5 months and remained in that state until termi-
nation of the experiment at six months. All female rats remained normotensive as did males at
the 500-ppm dose level. PRA was found to be significantly reduced in the 100-ppm treatment
males and normal in the 500-ppm treatment groups of both males and females. Dose-dependent
decreases in AII/PRA ratios and renal renin content were also observed. Pulmonary All con-
verting enzyme was not significantly altered. It was concluded that, since the observed
hypertension in the 100-ppm group of males was actually associated with reduction of PRA and
All, the renin-angiotensin system was probably not directly involved in this effect.
Webb et al. (1981) examined the vascular responsiveness of helical strips of tail
arteries in rats exposed to drinking water containing 100 ppm lead for seven months. These
investigators found that the mild hypertension associated with this regimen was associated
with increased vascular responsiveness to cradrenergic agonists.
Male rats exposed to lead jji utero and prior to weaning indirectly by their dams' drink-
ing water containing 0, 5, or 25 ppm lead as lead acetate, followed by direct exposure at the
same levels for five months (Victery et al., 1982b), showed no change in systolic blood pres-
sure. Rats exposed to the 25 ppm dose showed a significant (p <0.05) decrease in basal PRA.
Stimulation of renin release by administration of polyethylene glycol showed a significant
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increase in PRA but low All values. These yielded a significant (p <0.001) decrease in the
AII/PRA ratio. Basal renal renin concentrations were found to be significantly reduced in
both the 5 ppm (p <0.05) and 25 ppm (p <0.01) dose groups relative to controls.
Victery et al. (1983) exposed rats jjn utero to lead by maternal administration of 0, 5,
25, 100, or 500 ppm lead as lead acetate. The animals were continued on their respective dose
levels through one month of age. All exposure groups had PRA values significantly (p <0.05)
elevated relative to controls. Renal renin concentration was found to be similar to controls
in the 5 and 25 ppm groups but significantly increased (p <0.05) in the 100 and 500 ppm
groups. The plasma AII/PRA ratio was similar to controls in the 100 ppm group but was signi-
ficantly reduced (p <0.05) in the 500 ppm group.
It appears from the above studies that lead exposure at even low dose levels is capable
of producing marked changes in the renin-angiotension system and that the direction and mag-
nitude of these changes is mediated by a number of factors, including dose level, age, and sex
of the species tested, as well as dietary sodium content. Lead also appears capable of
directly altering vascular responsiveness to ct-adrenergic agents. The mild hypertension ob-
served with chronic low-level lead exposure appears to stem in part from this effect and not
from changes in the renin-angiotensin system. (See also Section 12.9.1 and the Addendum to
this document for a discussion of other work on the hypertensive effects of lead.)
12.5.6.6 Lead Effects on Uric Acid Metabolism. A report by Mahaffey et al. (1981) on rats
exposed concurrently to lead, cadmium, and arsenic alone or in combination found significantly
(p <0.05) increased serum concentrations of uric acid in the lead-only group. While the bio-
chemical mechanism of this effect is not clear, these data support certain observations in
humans concerning hyperuricemia as a result of lead exposure (see Section 12.5.3) and, also,
confirm an earlier report by Goyer (1971) showing increased serum uric acid concentration in
rats exposed to 1 percent lead acetate in drinking water for 84 weeks.
12.5.6.7 Lead Effects on Kidney Vitamin D Metabolism. Smith et al. (1981) fed rats vitamin
D-deficient diets containing either low or normal calcium or phosphate for two weeks. The
animals were subsequently given the same diets supplemented with 0.82 percent lead as lead
acetate. Ingestion of lead at this dose level significantly reduced plasma levels of 1,25
dihydrocholecalciferol in cholecalciferol-treated rats and in rats fed either a low phospho-
rous or low calcium diet while it had no effect in rats fed either a high calcium or normal
phosphorous diet. These data suggest decreased production of 1,25-dihydrocholecalciferol in
the kidney in response to lead exposure in concert with dietary deficiencies. These and
other data concerning lead effects on Vitamin-D metabolism were earlier discussed in detail
in Section 12.3.
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12.5.7 General Summary: Comparison of Lead's Effects on Kidneys in Humans and Animal Model*
It has been known for more than a century that kidney disease can result from lead
poisoning. Identifying the contributing causes and mechanisms of lead-induced nephropathy has
been difficult, however, in part because of the complexities of human exposure to lead and
other nephrotoxic agents. Nevertheless, it is possible to estimate at least roughly the range
of lead exposure associated with detectable renal dysfunction in both human adults and
children. Numerous studies of occupationally exposed workers have provided evidence for lead-
induced chronic nephropathy being associated with blood lead levels ranging from 40 to more
than 100 ug/dl, and some are suggestive of renal effects possibly occurring even at levels as
low as 30 pg/dl. In children, the relatively sparse evidence available points to the manifes-
tation of renal dysfunction only at quite high blood lead levels (usually exceeding 120
ug/dl). The current lack of evidence for renal dysfunction at lower blood lead levels 1n
children may simply reflect the greater clinical concern with neurotoxic effects of lead in-
toxication in children. The persistence of lead-induced renal dysfunction in children also
remains to be more fully investigated, although a few studies indicate that children diagnosed
as being acutely lead poisoned experience lead nephropathy effects lasting throughout adult-
hood.
Parallel results from experimental animal studies reinforce the findings in humans and
help illuminate the mechanisms underlying such effects. For example, a number of transient
effects in human and animal renal function are consistent with experimental findings of rever-
sible lesions such as nuclear inclusion bodies, cytomegaly, swollen mitochondria, and
increased numbers of iron-containing lysosomes in proximal tubule cells. Irreversible lesions
such as interstitial fibrosis are also well documented in both humans and animals following
chronic exposure to high doses of lead. Functional renal changes observed in humans have also
been confirmed in animal model systems with respect to increased excretion of amino acids and
elevated serum urea nitrogen and uric acid concentrations. The inhibitory effects of lead
exposure on renal blood flow and glomerular filtration rate are currently less clear in exper-
imental model systems; further research is needed to clarify the effects of lead on these
functional parameters in animals. Similarly, while lead-induced perturbation of the renin-
angiotensin system has been demonstrated in experimental animal models, further research is
needed to clarify the exact relationships among lead exposure (particularly chronic low-level
exposure), alteration of the renin-angiotensin system, and hypertension in both humans and
animals.
On the biochemical level, it appears that lead exposure produces changes at a number of
sites. Inhibition of membrane marker enzymes, decreased mitochondrial respiratory function/
cellular energy production, inhibition of renal heme biosynthesis, and altered nucleic acid
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synthesis are the most marked changes to have been reported. The extent to which these mito-
chondria! alterations occur is probably mediated in part by the intracellular bioavailability
of lead, which is determined by its binding to high affinity kidney cytosolic proteins and
deposition within intranuclear inclusion bodies.
Among the questions remaining to be answered more definitively about the effects of lead
on the kidneys is the lowest blood lead level at which renal effects occur. In this regard it
should be noted that recent studies in humans have indicated that the EDTA lead-mobilization
test is the most reliable technique for detecting persons at risk for chronic nephropathy;
blood lead measurements are a less satisfactory indicator because they may not accurately
reflect cumulative absorption some time after exposure to lead has terminated. Other ques-
tions include the following: Can a distinctive lead-induced renal lesion be identified either
in functional or histologic terms? What biologic measurements are most reliable for the pre-
diction of lead-induced nephropathy? What is the incidence of lead nephropathy in the general
population as well as among specifically defined subgroups with varying exposure? What is the
natural history of treated and untreated lead nephropathy? What is the mechanism of lead-
induced hypertension and renal injury? What are the contributions of environmental and genet-
ic factors to the appearance of renal injury due to lead? Conversely, the most difficult
question of all may well be to determine the contribution of low levels of lead exposure to
renal disease of non-lead etiologies.
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12.6 EFFECTS OF LEAD ON REPRODUCTION AND DEVELOPMENT
Studies of humans and animals indicate that lead may exert gametotoxic, embryotoxic, and
teratogenic effects that could influence the survival and development of the fetus and new-
born. It appears that prenatal viability and development may also be indirectly affected by
lead through its effects on the health of the expectant mother. The vulnerability of the con-
ceptus to such effects has contributed to concern that the unborn may constitute a group at
risk for the effects of lead on health. Also, certain information regarding male reproductive
functions has led to concern regarding the impact of lead on men.
12.6.1 Human Studies
12.6.1.1 Historical Evidence. Findings suggesting that lead exerts adverse effects on human
reproductive functions have existed in the literature since before the turn of the century.
For example, Paul (1860) observed that severely lead-poisoned pregnant women were likely to
abort, while those less severely intoxicated were more likely to deliver stillborn infants.
Legge (1901), in summarizing the reports of 11 English factory inspectors, found that of 212
pregnancies in 77 female lead workers, only 61 viable children were produced. Fifteen workers
never became pregnant; 21 stillbirths and 90 miscarriages occurred. Of 101 children born, 40
died in the first year. Legge also noted that when lead was fed to pregnant animals, they
typically aborted. He concluded that maternal exposure to lead resulted in a direct action of
the element on the fetus.
Four years later, Hall and Cantab (1905) discussed the increasing use of lead in nostrums
sold as abortifacients in Britain. Nine previous reports of the use of diachylon ("lead plas-
ter") in attempts to cause miscarriage were cited, along with 30 further cases of known or
apparent use of lead in attempts to terminate real or suspected pregnancy. Of 22 cases de-
scribed in detail, 12 resulted in miscarriage and all 12 exhibited marked signs of plumbism,
including a blue gum line. In eight of these cases, the women were known to have attempted to
induce abortion. Hall and Cantab1s report was soon followed by those of Cadman (1905) and
Eales (1905), who described three more women who miscarried following consumption of lead-
containing pills.
Oliver (1911) then published statistics on the effect of lead on pregnancy in Britain
(Table 12-13). These figures showed that the miscarriage rate was elevated among women em-
ployed in industries in which they were exposed to lead. Lead compounds were said by Taussig
(1936) to be known for their embryotoxic properties and their use to induce abortion. In a
more recent study by Lane (1949), women exposed to air lead levels of 750 M9/I"3 were examined
for effects on reproduction. Longitudinal data on 15 pregnancies indicated an increase in the
number of stillbirths and abortions. No data were given on urinary lead in these women, but
men in this sample had urinary levels of 75-100 pg/liter.
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TABLE 12-13. STATISTICS ON THE EFFECT OF LEAD ON PREGNANCY
Number of Number of
abortions and neonatal deaths
stillbirths per (first year) per
Sample 1000 females 1000 females
Housewives 43.2 150
Female workers (mill work) 47.6 214
Females exposed to lead premaritally 86.0 157
Females exposed to lead after marriage 133.5 271
Source: Oliver (1911).
The above studies clearly indicate an adverse effect of lead at high levels on human re-
productive functions, particularly miscarriages and stillbirths, when women are exposed to
lead during pregnancy. Although the mechanisms underlying these effects are unknown at this
time, many factors could contribute to such results. These factors range from indirect
effects of lead on maternal nutrition or hormonal state before or during pregnancy to more
direct gametotoxic, embryotoxic, fetotoxic, or teratogenic effects that could affect parental
fertility or offspring viability during gestation. Pregnancy is a stress that may place a
woman at higher risk for lead toxicity, because both iron deficiency and calcium deficiency
increase susceptibility to lead, and women have an increased risk of both deficiencies during
pregnancy and postparturition (Rom, 1976).
Early studies from the turn of the century generally suffer from methodological inadequa-
cies. They must be mentioned, however, because they provide evidence that effects of lead on
reproduction occurred at times when women were exposed to high levels of lead. Nevertheless,
evidence for adverse reproductive outcomes in women with obvious lead poisoning is of little
help in defining the effects of lead at much lower exposure levels. Efforts have been made to
define more precisely the points at which lead may affect reproductive functions in both the
human female and male, as well as in animals, as reviewed below.
12.6.1.2 Effects of Lead Exposure on Reproduction
12.6.1.2.1 Effects associated with exposure of women to lead. Since the time of the above
reports, women have been largely, though not entirely (Khera et al., 1980), excluded from oc-
cupational exposure to lead; also, lead is no longer used to induce abortion. Thus, little
new information is available on reproductive effects of chronic exposure of women to lead.
Various reports (Pearl and Boxt, 1980; Qazi et al., 1980; Timpo et al. , 1979; Singh et al. ,
1978; Angle and Mclntire, 1964) suggest that relatively high prenatal lead exposures do not
invariably result in abortion or in major problems readily detectable in the first few years
of life.
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These findings are based on only a few case histories, however, and are obviously not an ade-
quate sample. The data are confounded by numerous variables, and longer follow-ups are
needed.
In a sample population exposed to lead as well as other toxic agents from the Rbnnskar
smelter in Sweden, Nordstrom et al. (1978b) found an increased frequency of spontaneous
abortions among women living closest to the smelter. In addition to the exposure to multiple
environmental toxins, however, the study was confounded by failure to match exposed and
control populations for socioeconomic status, which could also bear upon the women's health.
A further study by the same authors (Nordstrom et al., 1979a) determined that female workers
at the Rb'nnskar smelter had an increased frequency of spontaneous miscarriage when the mother
was employed by the smelter during pregnancy or had been so employed prior to pregnancy and
still lived near the smelter. Also, women who worked in more highly polluted areas of the
smelter were more likely to have aborted than were other employees. This report, however,
suffers from the same deficiencies as the earlier study.
With regard to potential effects of lead on ovarian function in human females, Panova
(1972) reported a study of 140 women working in a printing plant for less than one year (1-12
months) where ambient air levels were under 7 ug Pb/m3. Using a classification of various age
groups (20-25, 26-35, and 36-40 yr) and type of ovarian cycle (normal, anovular, and disturbed
lutein phase), Panova claimed that statistically significant differences existed between the
lead-exposed and control groups in the age range 20-25 years. Panova1s report, however, does
not show the age distribution, the level of significance, or data on the specificity of her
method for classification. Zielhuis and Wibowo (1976), in a critical review of the above
study, concluded that the study design and presentation of data were such that it is difficult
to evaluate the author's conclusions. It should also be noted that no consideration was given
to the dust levels of lead, an important factor in print shops.
No other information is available for assessing the effects of lead on human ovarian
function or other factors affecting female fertility. Studies offering firm data on maternal
variables, e.g., hormonal state, that are known to affect the ability of the pregnant woman to
carry the fetus full term are also lacking.
12.6.1.2.2 Effects associated with exposure of men to lead. Lead-induced effects on male re-
productive functions have been reported in several instances. Among the earliest of these was
the review of Stofen (1974), who described data from the work of Neskov in the USSR involving
66 workers exposed chiefly to lead-containing gasoline (organic lead). In 58 men there was a
decrease or disappearance of erection, in 41 there was early ejaculation, and in 44 there was
a diminished number of spermatocytes. These results were confounded, however, by the presence
of the other constituents of gasoline.
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Lancranjan et al. (1975) reported lead-related interference with male reproductive func-
tions. A group of 150 workmen who had long-term exposure to lead in varying degrees was
studied. Clinical and toxicological criteria were used to categorize the men into four
groups: lead-poisoned workmen (mean blood lead level = 74.5 ug/dl) and those showing moderate
(52.8 ug/dl), slight (41 ug/dl), or "physiologic" (23 ugAH) exposure to lead. Moderately
increased lead absorption (52.8 M9/dl) was said to result in gonadal impairment. The effects
on the testes were believed to be direct, in that tests for impaired hypothalamopituitary
influence were negative. Also, semen analysis revealed asthenospermia and hypospermia in all
groups except those with "physiologic" absorption levels, and increased teratospermia was seen
in the two highest lead exposure groups.
An apparently exposure-related increase in erectile dysfunction was also found by
Lancranjan et al. (1975). Problems with ejaculation and libido were said to be more common in
the lead-exposed groups, but the incidence of such problems did not seem to be dose-dependent.
The frequency of these problems in a control group was invariably lower than in the lead-
exposed groups, however, so the lack of a clear-cut dose-response relationship may have merely
been due to inappropriate assignment of individuals to the high, moderate, and low-exposure
groups.
The Lancranjan et al. (1975) study has been criticized by Zielhuis and Wibowo (1976), who
stated that the distributions of blood lead levels appeared to be skewed and that exposure
groups overlapped in terms of lead intake. Thus, the means for each putative exposure group
may not have been representative of the individuals within a group. It is difficult to dis-
cern, however, if the men were improperly assigned to exposure level groups, as blood lead
levels may have varied considerably on a short-term basis. Zielhuis and Wibowo also stated
that the measured urinary ALA levels were unrealistically high for individuals with the stated
blood lead levels. This suggests that if the ALA values were correct, the blood lead levels
may have been underestimated. Other deficiencies of the study include the failure to use
matched controls and the exclusion of different proportions of individuals per exposure group
for the semen analyses.
Plechaty et al. (1977) measured lead concentrations in the semen of 21 healthy men.
Semen lead levels were generally less than blood lead levels, and no correlation was found be-
tween lead content of the semen and sperm counts or blood lead levels in this small sample.
Hypothalamic-pituitary-testicular relationships were investigated by Braunstein et al.
(1978) in men occupationally exposed at a lead smelter. Six subjects had 2-11 years of expo-
sure to lead and exhibited marked symptoms of lead toxicity. All had received one or more
courses of EDTA chelation therapy. This group was referred to as "lead-poisoned" (LP). Four
men from the same smelter had no signs of lead toxicity, but had been exposed for 1-23 years
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and were designated "lead-exposed" (LE). The control (C) group consisted of nine volunteers
whose socioeconomic status was similar to the lead workers. Mean (± standard error) blood
lead levels for the LP, LE, and C groups were 38.7 (± 3), 29.0 (± 5), and 16.1 (± 1.7) ug/dl,
respectively, at the time of the study. Previously, however, the LP and LE groups had exhi-
bited values as high as 88.2 (± 4) and 80 (± 0) |jg/dl, respectively. All three groups were
chelated and 24-hour urinary lead excretion values were 999 (± 141), 332 (± 17), and 225
(± 31) |jg for the LP, LE, and C groups, respectively. Frequency of intercourse was signifi-
cantly less in both lead-exposed groups than in controls. Sperm concentrations in semen of
the LP and LE men ranged from normal to severely oligospermic, and one from the LP group was
unable to ejaculate. Testicular biopsies were performed on "the two most severely lead-
poisoned men," one with aspermia and one with testicular pain. Both men showed increased
peritubular fibrosis, decreased spermatogenesis, and Sertoli cell vacuolization. The two lead
groups exhibited reduced basal serum testosterone levels, but displayed a normal increase in
serum testosterone following stimulation with human chorionic gonadotrophin. A similar rise
in serum follicle-stimulating hormone was seen following treatment with clomiphene citrate or
gonadotrophin-releasing hormone, although the LP men exhibited a lower-than-expected increase
in luteinizing hormone (LH). The LE men also appeared to have a reduced LH response, but the
decrease was not significant.
The results of the Braunstein et al. (1978) study suggest that lead exposure at high
levels may result in a defect in regulation of LH secretion at the hypothalamic-pituitary
level. They also indicate a likely direct effect on the testes, resulting in oligospermia and
peritubular fibrosis. However, the number of subjects in the study was quite small, and there
is also a possibility that the observed effects were precipitated by the EDTA chelation
therapy.
More recently, Wildt et al. (1983) compared two groups of men exposed to lead in a
Swedish battery factory. The high-lead men had had blood lead levels ^50 ug/dl at least once
prior to the study, while the "controls" seldom exceeded 30 ug/dl. There were two test
periods, in the fall and the following spring. For the first test, 14 men in the high-lead
group and 23 in the control group were examined; 16 were in each group for the second test.
Fourteen and 15 of these men from the high-lead and control groups, respectively, took part in
both tests. Blood lead values were obtained periodically over a six-month period. For the
two high-lead groups, blood lead values were 46.1 and 44.6 ug/dl, respectively (range: 25-75);
corresponding values for the controls were 31.1 and 21.5 ug/dl (range: 8-39). The high-lead
men tended to exhibit decreased function of the prostate and/or seminal vesicles, as measured
by seminal plasma constituents (fructose, acid phosphatase, magnesium, and zinc); however, a
significant difference was seen only in the case of zinc. More men in the high-lead than in
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the control group had low semen volumes, but the number of subjects did not allow a reliable
statistical analysis. The heads of sperm of high-lead individuals were more likely to swell
when exposed to a detergent solution, sodium dodecyl sulfate (SDS), which constituted a test
of functional maturity, but the results were still in the normal range. Conversely, the
leakage of lactate dehydrogenase isoenzyme X (LDH-X) was greater in control semen samples.
The values for live and motile sperm were lower in the control group. The data were
skewed, however, by the presence of some of the same men with low values in the control groups
for both sampling times. Another confounding factor was the fact that the high-lead and
control groups differed in a significant way: ten of the control men had current or past
urogenital tract infections versus none in the high-lead group, possibly explaining the inci-
dence of control samples with lowered sperm motility and viability. The observed decrease in
SDS resistance in the sperm of high-lead-group men may have been related to their apparent ab-
normal prostatic function or to an effect of lead on sperm maturation. In evaluating the
above results of Wildt et al. (1983) it must also be noted that even the "controls" had ele-
vated blood lead levels.
When Smith et al. (1983) compared blood lead levels in a sample of 80 infertile and 38
fertile men, no differences were seen between the two groups. Also, in a sample of 15 normal
and 16 vasectomized men, Butrimovitz et al. (1983) found no relationship between seminal lead
and sperm count or motility. Lead levels were relatively low (3.8 and 4.1 ug/dl in the intact
and vasectomized males, respectively), however, and such a result is not unexpected. At much
higher levels (66-139 ug/dl), five of seven lead-poisoned men examined by Cullen et al. (1984)
showed abnormal spermatogenesis, particularly oligospermia and azoospermia. Chelation therapy
produced only partial improvement in these patients.
12.6.1.3 Placental Transfer of Lead. The transfer of lead across the human placenta and the
consequent potential threat to the conceptus have been recognized for more than a century
(Paul, 1860). Documentation of placental transfer of lead to the fetus and data on resulting
fetal blood lead levels suggest a potential, but as yet not clearly defined, threat of subtle
embryotoxicity or other deleterious health effects.
The placental transfer of lead has been established, in part, by various studies that
have disclosed measurable quantities of lead in human fetuses or newborns, as well as off-
spring of experimental animals. The relevant data on prenatal lead absorption have been re-
viewed in Chapter 10, Section 10.2.4 of this document, and thus work dealing only with lead
levels will not be discussed further here.
12.6.1.4 Effects of Lead on the Developing Human
12.6.1.4.1 Effects of lead exposure on fetal metabolism. Prenatal exposure of the conceptus
to lead, even in the absence of overt teratogenicity, may be associated with biochemical
effects. This is suggested by studies relating fetal and cord-blood levels to changes in
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enzymes and precursors related to fetal heme synthesis. Haas et al. (1972) examined 294
mother-infant pairs for blood lead and urinary ALA levels. The maternal blood lead mean was
16.89 M9/dl, while the fetal blood lead mean was 14.98 pg/dl, with a correlation coefficient
of 0.54 (p <0.001). In the infants, blood lead levels and urinary ALA were positively cor-
related (r = 0.19, p <0.01), although the data were based on spot urines (which tends to limit
their value). The full biological significance of the elevated ALA levels is not clear, but
the positive correlation between lead in blood and urinary ALA for the group as a whole indi-
cates increased potential for impairment of heme synthesis at relatively low blood lead levels
in the fetus or newborn infant.
Subsequently, Kuhnert et al. (1977) measured ALA-D activity and levels of erythrocyte
lead in pregnant urban women and their newborn offspring. Cord erythrocyte lead levels ranged
from 16 to 67 ug/dl of cells, with a mean of 32.9. Lead levels were inversely correlated with
ALA-D activity (r = -0.58, p <0.01), suggesting that typical urban lead exposures could affect
fetal enzyme activity. Note, however, that ALA-D activity is related to blood cell age and is
highest in the younger cells. Thus, results obtained with cord blood, with its high percen-
tage of immature cells, are not directly comparable to those obtained with adult blood. In a
later study, Lauwerys et al. (1978) found no lead-related increase in erythrocyte porphyrin
levels in 500 mothers or their offspring. They did, however, report negative correlations
between ALA-D activity and blood lead levels in both mothers and their newborns. Maternal
blood lead levels averaged 10.2 |ag/dl, with a range of 3.1-31 ug/dl; corresponding values for
the newborns were 8.4 H9/dl and 2.7-27.3 M9/dl- Such results indicate that ALA-D activity may
be a more sensitive indicator of fetal lead toxicity than erythrocyte porphyrin or urinary ALA
levels.
12.6.1.4.2 Other toxic effects of intrauterine lead exposure. Fahim et al. (1976), in a
study on maternal and cord-blood lead levels, determined blood lead values in women having
preterm delivery and premature membrane rupture. Such women residing in a "lead belt" (mining
and smelting area) had significantly higher blood lead levels than women from the same area
delivering at full term. Fahim et al. (1976) also noted that among 249 pregnant women in a
control group outside the lead belt area, the percentages of women having preterm deliveries
and premature rupture were 3.0 and 0.4, respectively, whereas corresponding values for the
lead area (n = 253) were 13.04 and 16.99, respectively. A confusing aspect of this study,
however, is the similarity of blood lead levels in women from the presumptive low-lead and
lead belt areas. In fact, no evidence was presented that women in the lead belt group had ac-
tually received a greater degree of lead exposure during pregnancy than did control individ-
uals. Also, questions exist regarding analytical aspects of this study. Specifically, other
workers (e.g., see summary table in Clark, 1977) have typically found blood lead levels in
mothers and their newborn offspring to be much more similar than those of Fahim et al. (1976).
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In another study, Clark (1977) detected no effects of prenatal lead exposure in newborns
with regard to birth weight, hemoglobin, or hematocrit. He compared children born of 122
mothers living near a Zambian lead mine with 31 controls from another area. Maternal and in-
fant blood lead levels for the mine area were 41.2 (± 14.4) and 37.9 (± 15.3) ug/dl, respec-
tively. Corresponding values for control mothers and offspring were 14.7 (± 7.5) and 11.8 (±
5.6) jig/dl.
Nordstrb'm et al. (1979b) examined birth weight records for offspring of female employees
of a Swedish smelter and found decreased birth weights related to the following: (1) employ-
ment of the mothers at the smelter during pregnancy; (2) distance that the mothers lived from
the smelter; and (3) proximity of the mother's job to the actual smelting process. Similar
results were also seen for children born to mothers merely living near the smelter (Nordstrb'm
et al., 1978a). Nordstrom et al. (1979b) also investigated birth defects in offspring of the
female smelter workers and in populations living at various distances from the smelter. They
concluded that the frequencies of both single and multiple malformations were increased when
the mother worked at the smelter during pregnancy.
The number of smelter workers with malformed offspring was relatively small (39 of 1291).
The incidence of children with birth defects whose mothers worked while pregnant was 5.8 per-
cent (17 of 291). Five of the six offspring with multiple malformations were in this group,
suggesting that the observed effect may have been a real one. Nevertheless, a crucial factor
in evaluating all of these results is the exposure of workers and the nearby population to a
number of toxic substances, including not only lead, but arsenic, mercury, cadmium, and sulfur
dioxide as well.
Alexander and Delves (1981) found that the mean blood lead concentrations of pregnant and
non-pregnant women living in an urban area of England were approximately 4 ug/dl higher than
those for similar groups living in a rural area. The mean concentrations for the urban and
rural pregnant women were 15.9 and 11.9 ug/dl, respectively (p <0.001), but there were no
demonstrable effects of the higher maternal blood lead levels on any aspect of perinatal
health. The rate for congenital abnormality was higher in the rural area, suggesting that
whatever the cause, it was unlikely to be related to maternal levels of lead.
Khera et al. (1980) measured placental and stillbirth tissue lead in occupationally
exposed women in the United Kingdom. Regardless of the incidence of stillbirths, placental
lead concentrations were found to increase with duration of occupational exposure, from 0.29
ug/g at <1 year exposure to 0.48 ug/g at >6 years exposure for a group of 26 women aged 20-29
years. Placental lead concentrations also increased with age of the mother, independently of
time of occupational exposure, and ranged from 0.30 (± 0.16) ug/g for those <20 years old to
0.51 (±0.44) ug/g for those £30 years old. Average placental lead concentrations for 20 occu-
pationally exposed women whose babies were stillborn were higher [0.45 (± 0.32) ug/g] than the
12-199
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average level [0.29 (± 0.09) |jg/g] for placentas from eight mothers who had not been occupa-
tional ly exposed for at least two years. The authors noted, however, that it was not possible
to say whether occupational exposure caused any of the stillbirths or whether the high lead
levels were merely consequential to the fetal death. Interpretation of this study is also
somewhat complicated by the fact that the average placental lead concentration was about one-
third that reported earlier by this group (Wibberley et al., 1977). These differences were
attributed to methodological changes and to changes in concentration during storage of placen-
tas at -20°C (Khera et al., 1980).
A study by Roels et al. (1978b) reported placental lead values of 0.08 (± 0.05) ug/g
(range = 0.01-0.40 pg/g) from a variety of locations in Belgium, but these data indicated no
correlation between lead concentration and birth weight. In contrast, placental lead has been
reported to be associated with decreased activity of a placental enzyme, steroid sulfatase
(Karp and Robertson, 1977). A similar association was found for mercury, suggesting that
either metal or both together could have affected the enzyme activity or that the authors had
merely uncovered a spurious correlation. There is also some evidence that lead levels in bone
samples from stillborn children are higher than would otherwise be expected (Khera et al.,
1980; Bryce-Smith et al., 1977), but the data are inconclusive. (See the Addendum to this
document for a discussion of more recent evidence concerning possible effects of intrauterine
lead exposure on prenatal and postnatal development in children.)
12.6.1.4.3 Paternally mediated effects of lead. There is increasing evidence that exposure
of male laboratory animals to toxic agents can result in adverse effects on their offspring,
including decreased litter size, birth weight, and survival (see Section 12.6.2.2.1). Mutage-
nic effects are the most likely cause of such results, but other mechanisms have been proposed
(Soyka and Joffe, 1980). In the following cases, exposure of human males to lead has been im-
plicated as the cause of adverse effects on the conceptus.
According to Koinuma (1926) in a brief report, 24.7 percent of workmen exposed to lead in
a storage battery plant had childless marriages, while the value for men not so exposed was
14.8 percent. Rates for miscarriages or stillbirths in wives of lead-exposed men and controls
were 8.2 and 2.8 percent, respectively, while corresponding figures for neonatal deaths were
24.2 and 19.2 percent. The comparisons were based on 170 lead-exposed and 128 control men.
These differences in fertility and prenatal mortality, while not dramatic, are suggestive of a
male-mediated lead effect; however, the reliability of the methodology used in this study can-
not be determined, due to the brevity of the report.
In a study of the pregnancies of 104 Japanese women before and after their husbands began
lead-smelter work, miscarriages increased to 8.3 percent of pregnancies from a pre-exposure
rate of 4.7 percent (Nogaki, 1957). The miscarriage rate for 75 women whose husbands were
12-200
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not occupationally exposed to lead was 5.8 percent. In addition, exposure to lead was
related to a significant increase in the ratio of male to female offspring at birth. Lead
content of paternal blood ranged from 11 to 51.7 pg/dl [mean = 25.4 (± 1.26) ug/dl], but was
not correlated with reproductive outcome, except in the case of the male-to-female offspring
ratio. The reported blood lead levels appear low, however, in view of the occupational expo-
sure of these men, and were similar to those given for controls [mean = 22.8 (± 1.63)
jjg/dl]. Also, maternal age and parity appear not to have been well controlled for in the
analysis of the data on reproductive outcome. Another report (Van Assen, 1958) on fatal birth
defects in children conceived during a period when their father was lead-poisoned (but neither
before nor after) also suggests but does not clearly demonstrate paternally-mediated effects
of lead.
In the study by Nordstrom et al. (1979b), women employed at the Rb'nnskar smelter in
Sweden were found to have higher miscarriage rates if their husbands were also employed at the
smelter. This was true only of their third or later pregnancies, however, suggesting that the
effect was related to long-term exposure of the male gametogenic stem cells. Whether this was
a lead effect or that of other toxins from the smelter is not clear.
12.6.1.5 Summary of the Human Data. The literature on the effects of lead on human reproduc-
tion and development leaves little doubt that lead can, at high exposure levels, exert signi-
ficant adverse health effects on reproductive functions. Most studies, however, only examined
the effects of prolonged moderate to high exposure to lead, such as that encountered in indus-
trial situations, and many reports do not provide definite information on exposure levels or
blood lead levels at which specific effects were observed. Also, the human data were largely
derived from studies involving relatively small numbers of individuals and therefore do not
allow for discriminating statistical analysis. These reports are often additionally confound-
ed by the failure to include appropriate controls and, in some cases, by the presence of addi-
tional toxic agents or disease states. These and other factors obviously make interpretation
of the data difficult. Based on the Lancranjan et al. (1975) and Wildt et al. (1983) studies,
it appears possible that effects on sperm or the testis may result from chronic exposure at
blood lead values of 40-50 ug/dl, but additional data are greatly needed. Exposure data
related to reproductive functions in the female are so lacking that even a rough estimate is
impossible. Data on maternal exposure levels at which effects may be seen in human fetuses or
infants are also quite meager or equivocal. However, the results of Haas et al. (1972),
Kuhnert et al. (1977), and Lauwerys et al. (1978) suggest possible perinatal effects on heme
metabolism at maternal blood levels considerably below 30 ug/dl. [More recent studies dealing
with the effects of relatively low-level lead exposure on development both prenatally (e.g.,
congenital anomalies, birth weight, gestational age) and postnatally (e.g., infant mental
development, child stature) are reviewed in an Addendum to this document.]
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12.6.2 Animal Studies
12.6.2.1 Effects of Lead on Reproduction
12.6.2.1.1 Effects of lead on male reproductive functions. Among the first investigators to
report infertility in male animals due to lead exposure were Puhac et al. (1963), who exposed
rats to lead via their diet. Ability to sire offspring returned, however, 45 days after ces-
sation of treatment. More recently, Varma et al. (1974) gave a solution of lead subacetate in
drinking water to male Swiss mice for four weeks (mean total intake of lead = 1.65 g). The
fertility of treated males was reduced by 50 percent. Varma and coworkers calculated the
mutagenicity index (number of early fetal deaths/total implants) to be 10.4 for lead-treated
mice versus 2.98 for controls (p <0.05). The major differences in fecundity appeared to have
been due to differing pregnancy rates, however, rather than prenatal mortality. Impairment of
male fertility by lead rather than lead-induced mutagenicity was thus likely to have been the
primary toxic effect observed. Additionally, it has been suggested by Leonard et al. (1973),
that effects seen following administration of lead acetate in water may be due to resulting
acidity, rather than to lead. Also, Eyden et al. (1978) found no decrease in fertility of
male mice fed 0.1 percent lead acetate in the diet for 64 weeks.
Several animal studies have found lead-associated damage to the testes or prostate,
generally at relatively high doses. Golubovich et al. (1968) found a decrease in normal sper-
matogonia in the testes of rats gavaged for 20 days with lead (2 mg/kg per day). Desquamation
of the germinal epithelium of the seminiferous tubules was also increased, as were degenera-
ting spermatogonia. Hilderbrand et al. (1973) also noted testicular damage in male rats given
oral lead (100 ug/day for 30 days). Egorova (cited in Stb'fen, 1974) injected lead at a dose
of 2 pg/kg six times over a ten-day period and reported testicular damage.
Ivanova-Chemishanska et al. (1980) investigated the effect of lead on male rats adminis-
tered 0.0001 or 0.01 percent solutions of lead acetate over a four-month period. The authors
reported that changes in enzymatic activity and in levels of disulfide and ATP were observed
in testicular homogenates. No histopathological changes in testicular tissue were found, but
the fertility index for treated males was decreased. Offspring of those males exhibited post-
partum "failure to thrive" and stunted growth. Such data suggest biological effects due to
chronic lead exposure of the male, but the study is difficult to evaluate due to limited in-
formation on the experimental methods, particularly the dose levels actually received.
In a more recent study of lead's effects on the male reproductive tract, Chowdhury et al.
(1984) found testicular atropy along with cellular degeneration in rats exposed to lead ace-
tate in water at 1 g/1 for 60 days. Blood lead level at that exposure averaged 142.6 ug/dl.
At a lower exposure level yielding a blood lead concentration of 71.7 ug/dl, seminiferous
tubular diameter was singificantly reduced, as was spermatid count. No significant changes
were seen at a blood lead level of 54.0 ug/dl.
12-202
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Non-rodent species have also been investigated. No histopathological changes were seen
during an examination of the testes of rabbits (Willems et al., 1982). Five males per group
were dosed subcutaneously with up to 0.5 mg/kg lead acetate three times weekly for 14 weeks.
Blood lead levels at termination of treatment were 6.6 and 61.5 pg/dl for control and high-
dose rabbits, respectively.
Lead-related effects on spermatozoa have also been reported. For example, Stowe et al.
(1973) described the results of a low-calcium and phosphate diet containing 100 ppm lead (as
acetate) fed to dogs from 6 to 18 weeks of age. This dose resulted in a number of signs of
toxicity, including spermatogonia with hydropic degeneration. In a study by Mai sin et al.
(1975), male mice received up to 1 percent lead in the diet, and the percentage of abnormal
spermatozoa increased with increasing lead exposure. Eyden et al. (1978) also fed 1 percent
lead acetate in the diet to male mice. By the eighth week, abnormal sperm had increased;
however, the affected mice showed weight loss and other signs of general toxicity. Thus, the
effect on spermatogenesis was not indicative of differential sensitivity of the gonad to lead.
Krasovskii et al. (1979) observed declines in motility, duration of motility, and osmotic
stability of sperm from rats given 0.05 mg/kg lead orally for 20-30 days. Damage to gonadal
blood vessels and to Leydig cells was also seen. Rats treated for 6-12 months exhibited ab-
normal sperm morphology and decreased spermatogenesis. In the report of Will ems et al. (1982)
described above, however, no effects on sperm count or morphology were seen in rabbits.
Lead acetate effects on sperm morphology were also tested in mice given about one six-
teenth to one half an LD50 dose by i.p. injection on five consecutive days (Bruce and Heddle,
1979; Wyrobek and Bruce, 1978; Heddle and Bruce, 1977). The two lowest doses (apparently 100
and 250 mg/kg) resulted in only a modest increase in morphologically abnormal sperm 35 days
after treatment, but the 500 or 900 mg/kg doses resulted in up to 21 percent abnormal sperm.
That lead could directly affect developing sperm or their cellular precursors is made
more plausible by the data of Timm and Schulz (1966), who found lead in the seminiferous
tubules of rats and in their sperm. The mechanisms for lead's effects on the male gonad or
gamete are unknown, although Golubovich et al. (1968) found altered RNA levels in the testes
of lead-exposed rats. They suggested that testicular damage was related to diminished riboso-
mal activity and inhibition of protein synthesis. As noted above, Ivanova-Chemishanska et al.
(1980) observed biochemical changes in testes of lead-treated mice. Nevertheless, such obser-
vations are only initial attempts to determine a mechanism for the observed effects of lead.
A more likely mechanism for such effects on the testes may be found in the work of Donovan et
al. (1980), who found that lead inhibited androgen binding by the cytosolic receptors of mouse
prostate. This could provide a mechanism for the observation of Khare et al. (1978), who
found that injection of lead acetate into the rat prostate resulted in decreased prostatic
weight; no such changes were seen in other accessory sex glands or in the testes.
12-203
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Effects on hormonal production or on hormone receptors could also explain the results of
Maker et al. (1975), who observed a delay in testicular development and an increase in age of
first mating in male mice of two strains whose dams were given 0.08 percent lead (C57B1/6J) or
0.5 percent lead (Swiss-Webster albino) during pregnancy and lactation. The weanling males
were fed these same doses in their diets through 60 days of age.
In attempts to further examine the possible mechanisms of effects on the male, Wiebe et
al. (1982) treated rats with lead acetate injected s.c. from gestation day 9 every 3-4 days
throughout pregnancy and for the first 2-3 weeks of lactation. Testes from the two- to three-
week-old male offspring of treated mothers had normal weights and seminiferous tubule diame-
ters, but yielded testicular homogenates with decreased ability to convert progesterone to a
variety of metabolites. Such results, in addition to direct enzyme assays, showed decreased
activities of 3a-, 3p-, and 20crhydroxysteroid oxidoreductases and of the 5crreductase and
enzymes. Receptor binding of FSH was also significantly reduced. More recently,
Wiebe et al. (1983) compared Sertoli cells isolated from prepubertal rats and cultured in the
presence of either the acetate salts of lead or sodium (2.64 x 10 M). After 24 hours, lead
exposure was associated with a 10-20 percent decrease in FSH binding and in the production of
cyclic AMP; at 96 hours, the decrease was 75 percent. Sixteen-day-old rats were more sensi-
tive than those 20 days of age and exhibited a 97 percent decrease in FSH- induced cyclic AMP
by 144 hours of lead exposure. The ability of Sertoli cells to metabolize progesterone and
their steroidogenic response to FSH was also inhibited by 48-hour lead exposure. Activity of
cellular 3p-hydroxysteroid dehydrogenase was decreased after lead exposure in Sertoli cells
and Tin vitro in the presence of PbCl2 in the assay buffer. These results support the concept
that lead may directly affect testicular enzymes or may act indirectly by a reduction in
testicular binding of FSH and production of cyclic AMP.
Another potential mechanism underlying lead's effects on sperm involves its affinity for
sulfhydryl groups. Mammalian sperm possess high concentrations of sulfhydryls believed to be
involved in the maintenance of motility and maturation via regulation of stability in sperm
heads and tails (Bedford and Calvin, 1974; Calvin and Bedford, 1971). It has also been found
that blockage of membrane thiols inhibits sperm maturation (Reyes et al., 1976).
12.6.2.1.2 Effects associated with exposure of females to lead. Numerous studies have
focused on lead exposure effects in females. For example, effects of lead on reproductive
functions of female rats were studied by Hilderbrand et al. (1973), using animals given lead
acetate orally at doses of 5 or 100 ug for 30 days. Control rats of both sexes had the same
blood lead levels. Blood lead levels of treated females were higher than those of similarly
treated males: 30 versus 19 ug/dl at the low dose, and 53 versus 30 ug/dl at the high dose.
The females exhibited irregular estrous cycles at both doses. When blood lead levels reached
50 ug/dl, they developed ovarian follicular cysts, with reductions in numbers of corporalutea.
12-204
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In a subsequent study (Der et al., 1974), lead acetate (100 pg lead per day) was injected
subcutaneously for 40 days in weanling female rats. Treated rats received a low-protein
(4 percent) or adequate-protein (20 percent) diet; controls were given the same diets without
lead. Females on the low-protein, high-lead diet did not display vaginal opening during the
treatment period and their ovaries decreased in weight. No estrous cycles were observed in
animals from either low-protein group; those of the adequate diet controls were normal, while
those of the rats given adequate protein plus lead were irregular in length. Endometrial pro-
liferation was also inhibited by lead treatment. Blood lead levels were 23 ug/dl in the two
control groups, while values for the adequate- and low-protein lead-treated groups were 61 and
1086 M9/dl, respectively. The reports of Hilderbrand et al. (1973) and Der et al. (1974)
suggest that lead chronically administered in high doses can interfere with sexual development
in rats and the body burden of lead is greatly increased by protein deprivation.
Maker et al. (1975) noted a delay in age at first conception in female mice of two
strains exposed to 0.08 percent (C57B1/6J) or 0.5 percent lead (Swiss-Webster) indirectly via
the maternal diet (while jji utero and nursing) and directly up to 60 days of age. These
females were retarded in growth and tended to conceive only after reaching weights approxi-
mating those at which untreated mice normally first conceive. Litters from females that had
themselves been developmentally exposed to at least 0.5 percent lead had lower survival rates
and retarded development. More recently, Grant et al. (1980) reported delayed vaginal opening
in rats whose mothers were given 25, 50, or 250 ppm lead (as lead acetate) in their drinking
water during gestation and lactation followed by equivalent exposure of the offspring after
weaning. The vaginal opening delays in the 25-ppm females occurred in the absence of any
growth retardation or other developmental delays and were associated with median blood lead
levels of 18-29 ug/dl.
Although most animal studies have used rodents, Vermande-Van Eck and Meigs (1960) admin-
istered lead chloride intravenously to female rhesus monkeys. The monkeys were given 10
ing/week for four weeks and 20 mg/week for the next seven months. Lead treatment resulted in
cessation of menstruation, loss of color of the "sex skin" (presumably due to decreased
estrogen production), and pathological changes in the ovaries. One to five months after lead
treatment ceased, menstrual periods resumed, the sex skin returned to a normal color, and the
ovaries regained their normal appearance. Thus, there was an apparent reversal of these
effects on female reproductive functions, although there were no confirmatory tests of
fertility.
The above studies indicate that pre- and/or postnatal exposure of female animals to lead
can affect pubertal progression and hypothalamic-pituitary-ovarian-uterine functions. The
observations of delayed vaginal opening may reflect delayed ovarian estrogen secretion due to
12-205
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toxicity to the ovary, hypothalamus, or pituitary. One study has demonstrated decreased
levels of circulating follicle-stimulating hormone (Petrusz et al., 1979), and others discus-
sed previously have shown lead-induced ovarian atrophy (Stowe and Goyer, 1971; Vermande-Van
Eck and Meigs, 1960), again suggesting toxicity involving the hypothalamic-pituitary-ovarian-
endometrial axis.
12.6.2.2 Effects of Lead on the Offspring. This section discusses developmental studies of
animals whose parents (one or both) were exposed to lead. Possible male-mediated effects as
well as effects of exposure during gestation are reviewed. Results obtained for offspring of
females given lead following implantation or throughout pregnancy are summarized in Tables
12-14 and 12-15.
12.6.2.2.1 Male-mediated effects. A few studies have focused on male-mediated lead effects
on the offspring and have suggested that paternally transmitted effects of lead may cause re-
ductions in litter size, offspring weight, and survival rate.
Cole and Bachhuber (1915), using rabbits, were the first to report paternal effects of
lead intoxication. In their study, the litters of dams sired by lead-intoxicated male rabbits
were smaller than those sired by controls. Weller (1915) similarly demonstrated reduced birth
weights and survival among offspring of lead-exposed male guinea pigs.
Offspring of lead-treated males from the Ivanova-Chemishanska et al. (1980) study de-
scribed above were affected in a variety of ways, e.g., they exhibited "failure to thrive" and
lower weights than did control progeny at one and three weeks postpartum. These results are
difficult to interpret, however, without more specific information on the experimental methods
and dosing procedures.
12.6.2.2.2 Results of lead exposure of both parents. Only a few studies have assessed the
effects of lead exposure of both parents on reproduction. Schroeder and Mitchener (1971)
found a reduction in the number of offspring of rats and mice given drinking water containing
25 ppm lead. According to the data of Schroeder et al. (1970), however, animals in the 1971
study may have been chromium-deficient, and the Schroeder and Mitchener (1971) results are in
marked contrast to those of an earlier study by Morris et al. (1938), who reported no signifi-
cant reduction in weaning percentage among offspring of rats fed 512 ppm lead.
In another study, Stowe and Goyer (1971) assessed the relative paternal and maternal
effects of lead as measured by effects on the progeny of lead-intoxicated rats. Female rats
fed diets with or without 1 percent lead were mated with normal males. The pregnant rats were
continued on their respective rations with or without lead throughout gestation and lactation.
Offspring of these matings, the Fj generation, were fed the rations of their dams and were
mated in combinations as follows: control female to control male (CF-CM), control female to
lead-intoxicated male (CF-PbM), lead-intoxicated female to control male (PbF-CM), and lead-
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TABLE 12-14. EFFECTS OF PRENATAL
STUDIES
EXPOSURE TO LEAD ON THE OFFSPRING OF LABORATORY AND DOMESTIC ANIMALS:
USING ORAL OR INHALATION ROUTES OF EXPOSURE
Treatment
Species Test agent Dose and node
Rat Lead acetate 512 ppm in diet
10,000 pp» in diet
39 mg/kg/day, po
390 ag/kg/day, po
255-478 «g/kg/day in
water
31.9-319 ppM in water
50*250 ppn in water
25 ppn in water
0.5-5 ppa in water
5 or 50 ppn in water
£ 31.9-47.8 •g/kg/day, po
^ 63.7 «g/kg/day, po
^ 150 ng/kg/day, po
500 ppn in water
5 ppm in water
5-500 ppm in water
Tetraethyl lead 1.6-3.2 «g/kg/day, po
0.064 ng/kg/day, po
0.64 ng/kg/day, po
6.4 mg/kg/day, po
Tetranethyl lead 10-28.7 mg/kg/day, po
Trinethyl lead 3.6-7.2 ng/kg/day, po
chloride
Lead nitrate 1 pp* in water
10 ppa in water
Lead (aerosol) 1 or 3 mg/m3, inhaled
10 ag/H3, inhaled
Tiningc
all
all
6-16
6-8
all, LAC
all
all, LAC
all, LAC
all, LAC
all, LAC
all
all
6-18
1-18 or 1-21
1-18 or 1-21
all, LAC
9-11 or 12-14
6-16
6-16
6-8
9-11 or 12-14
9-11 or 12-14
all
all
1-21
1-21
Effect on the offspring3
Mortality Fetotoxicity Malformation Reference
? Morris et al. (1938)
* + ? Stowe and Goyer (1971)
Kennedy et al. (1975)
? +d ? Murray et al. (1978)
±e + - Oilts and Ahokas (1979, 1980)
* - Kiwnel et al. (1980)
±
+ + - Herman et al. (1981)
Miller et al. (1982)
* - - Wardell et al. (1982)
+ - Hayashi (1983a)
+ - Hayashi (1983b)
+ - Victery et al. (1983)
± + - McClain and Becker (1972)
Kennedy et al. (1975)
± + - McClain and Becker (1972)
+
? Hubenmnt et al. (1976)
? -* ? Prigge and Greve (1977)
-------
TABLE 12-14. (continued)
o
oo
Species
House
Sheep
Treatment
Test agent Dose and mode
Lead acetate 3,185 ppm in diet
780-1,593 ppm in diet
3,135 ppn in diet
1,593-6,370 ppm in diet
1,595-3,185 ppm in diet
39 *g/kg/day, po
390 mg/kg/day, po
0.1-1.0 g/1 in water
637-3,185 ppm in diet
1,593 ppm in diet
3,185 ppn in diet
1,250 ppm in diet
3,185 ppm in diet
1,250 ppm in diet
2,500-5,000 ppm
in diet
1,250 ppm in diet
1,000 ppm in diet
Tetraethyl lead 0.06 mg/kg/day, po
0.64 mg/kg/day, po
6.4 mg/kg/day, po
Lead powder 0. 5- 16 -mg/kg/day, in
diet1
Timingc
1-7
1-16,17, or 18
1-16,17, or 18
1-15,16, or 17
7-16,17, or 18
5-15
5-7
all
1-18
1-16,17, or 18
1-16,17, or 18
all
1-16,17, or 18
all
all
all
all, LAC
6-16
6-16
6-8
all
Effect on the offspring8
Mortality Fetotoxicity Malformation
+ ± N/A
; :tf,j ;
7 <-'( 7
? + ?
+ + -
7 7
+ ? ?
: : :
+ + -
+ ?
Reference
Jacquet (1977)
Jacquet et al. (1977b)
Gerber and Maes (1978)
Gerber et al . (1978)
Kennedy et al . (1975)
Leonard et al. (1973)
Maisin et al. (1975)
Jacquet et al. (1975)
Jacquet (1976)
Talcott and Koller (1983)
Kennedy et al. (1975)
Shama and Buck (1976)
+ = present; - = effect not seen; ± = ambiguous effect; ? = effect not examined or insufficient data.
Oose as elemental lead; po = per os (gavage).
cSpecific gestation days when exposed; LAC = also during lactation.
Decreased numbers of dendritic spines and malformed spines at day 30 postpartun.
Litter size values for high-dose group suggestive of an effect.
ALA-D activity was decreased.
%ree tissue porphyrins increased in kidneys.
Henatocrit was decreased.
Vetal porphyrins were increased, except in the low-dose fetuses assayed on gestation day 18.
•'Decreased heme and fetal weight.
Incorporation of Fe into heme decreased, and growth was retarded.
Decreased placental blood flow.
-------
TABLE 12-15. EFFECTS OF PRENATAL LEAD EXPOSURE ON OFFSPRING OF LABORATORY ANIMALS:
RESULTS OF STUDIES EMPLOYING ADMINISTRATION OF LEAD BY INJECTION
Species
Rat
_
i
no
a
•£>
House
Treatment
Test agent Dose and node
Lead acetate 15.9 ngAg, ip
Lead nitrate 31.3 mg/kg, iv
31.3 mg/kg, iv
31.3 ng/kg, iv
3.13 mg/kg, iv
15.6 mg/kg, iv
15.6 ng/kg, iv
unknown, iv
31.3 mg/kg, iv
15.6 mg/kg, iv
5 ng/kg, iv
25 mg/kg, iv
Lead chloride 7.5 ng/kg^
75 Kg/kg,
Tri nethyl lead 20.2 mg/kg, iv
chloride 23.8 mg/kg, iv
Lead acetate 9.56-22.3 mg/kg, ip
9.56 mg/kg, ip
22.3 »g/kg, ip
22.3 mg/kg, ip
Lead chloride 29.8 «g/kg, iv
29.8 mg/kg, iv
Timingc
9
8
9 or 16
10-14,
15,17
9 or 15
9
15
8 or 9
17
17
9 or 15
9 or 15
9
9
12
9,10,13, or 15
8
9
9
10 or 12
3 or 4
6
Effect on the offspring3
Mortality Fetotoxicity Malformation Reference
+ + + Zegarska et al. (1974)
+ + McClain and Becker (1975)
Hackett et al. (1978, 1979)
+ 7 7
+ ? + Coro Antich and Aaoedo Non
(1980)
+ - Minsker et al. (1982)
- e Hackett et al. (1982a,b)
± - - Mclellan et al. (1974)
+9 + I
+ + Jacquet and Gerber (1979)
+ *
+ + +
+ ? ? Wide and Nilsson (1977)
+ N/A N/A
-------
TABLE 12-15. (continued)
Species
Hamster
*— •
IV)
i
IV)
o
Test agent
Lead acetate
Lead acetate or
chloride
Lead nitrate
Treatment
Dose and Mode
31.9 ag/kg, iv
31.9 or 37.3 «g/kg, iv
31.3 «g/kg. iv
15.6-31.3 mg/kg, iv
31.3 ng/kg, iv
31.3 ag/kg. iv
Tiihngc
8
8
7, 8. or 9
8 or 9
8
8
Effect on the offspring3
Mortality Fetotoxicity Malformation Reference
+ ? + Fen« (1969)
? ? + Fem and Carpenter (1967)
? ? + Fem and Carpenter (1967)
•f ? + Fem and Fern (1971)
+ + + Carpenter and Fen (1977)
+ +h + Gale (1978)
a+ = effect present; - = effect not seen; ± = aabiguous effect; ? = effect not examined or insufficient data.
Dose as elemental lead; ip = intraperitoneally; iv = intravenously.
cSpecific gestation days when exposed.
''with the exception of day 17.
eHo fetuses survived to be examined for malfon»ation.
No dosage route specified.
°0nly after day 10 treatment.
"Delayed ossification (fetal weights not given).
-------
intoxicated female to lead-intoxicated male (PbF-PbM). The results of this study are shown in
Table 12-16.
The paternal effects of lead included reductions of 15 percent in the number of pups per
litter, 12 percent in mean pup birth weight, and 18 percent in pup survival rate. The mater-
nal effects of lead included reductions of 26 percent in litter size, 19 percent in pup birth
weight, and 41 percent in pup survival. The combined male and female effects of lead toxicity
resulted in reductions of 35 percent in the number of pups per litter, 29 percent in pup birth
weight, and 67 percent in pup survival to weaning. Stowe and Goyer classified the effects of
lead upon reproduction as gametotoxic, intrauterine, and extrauterine. The gametotoxic ef-
fects of lead seemed to be irreversible and had additive male and female components. Intra-
uterine effects were presumed to be due to lead uptake by the conceptus, plus gametotoxic ef-
fects. The extrauterine effects were due to the passage of lead from the dam to the nursing
pups, adding to the gametotoxic and intrauterine effects.
Leonard et al. (1973), however, found no effect on the reproductive performance of groups
of 20 pairs of mice given lead in their drinking water over a nine-month period. Lead doses
ranged from 0.1 to 1.0 g/1. A total amount of 31 g/kg was ingested at the high dose, equiva-
lent to ingestion of 2.2 kg lead by a 70-kg man over the same time period.
More recently, rats from mothers that were exposed to lead at 5 or 50 ppm or to lead plus
cadmium at concentrations of 5 ppm Pb + 0.1 ppm Cd or 50 ppm Pb + 5 ppm Cd in the drinking
water during gestation and lactation were themselves continued on the same treatments (Herman
et al., 1981). All individuals were treated during mating, with the mated females also being
treated during gestation and given a teratological examination at day 20. Other females were
allowed to litter and treatment was continued through postpartum day 21. Treatment with lead
or lead plus cadmium appeared to cause preimplantation loss. In the groups allowed to litter,
maternal weight gain, litter size, and offspring survival and weight were all said to be re-
duced in both lead groups and more severely in the Pb plus Cd groups. Eye opening was also
delayed in all groups. The value of these results is not clear, however, as no statistical
analysis was mentioned.
12.6.2.2.3 Lead effects on implantation and early development. Numerous studies have been
performed to elucidate mechanisms by which lead causes prenatal death. They suggest two
mechanisms of action for lead, one on implantation and the other (mainly at higher doses) on
fetal development. The latter is discussed primarily in Section 12.6.2.2.4.5.
Mai sin et al. (1975) exposed female mice to dietary lead for 18 days after mating; the
number of both pregnancies and surviving embryos decreased. Similarly, exposure of female
mice to lead via their diet (0.125-1.0 percent) from mating to 16-18 days afterward (Jacquet,
1976; Jacquet et al., 1975) resulted in the following: decreased incidence of pregnancy and
number of corpora lutea; increased number of embryos dying after implantation at the highest
dosages; decreased body weights of surviving fetuses; and fatalities among treated dams at the
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TABLE 12-16. REPRODUCTIVE PERFORMANCE OF F! LEAD-INTOXICATED RATS (MEANS ± STANDARD ERRORS)
ro
t
ro
i—•
TN3
Parameter
Litters observed
Pups per litter
Pup birth weight, g
Weaned rats per litter
Survival rate, %
Litter birth weight, a.
Dam breeding weight
Litter birth weight, ^
Dam whelping weight
Gestational qain,
Pups per litter *•
Nonfetal gestational
gain per fetus, g
CF-CM
22
11.90
6.74
9.84
89.80
28.04
19.09
11.54
3.93
±
±
±
±
±
±
±
±
0.40
0.15
0.50
3.20
1.30
0.80
0.60
0.38
10.
5.
7.
73.
22.
15.
11.
4.
Type of
CF-PbM
24
10 ±
92 ±
04 ±
70 ±
30 ±
97 ±
20 ±
83 ±
0.50
0.13C
0.77C
7.90
0.90C
0.58C
0.74
0.47
mating
PbF-CM
36
8.78 ±
5.44 ±
5.41 ±
52.60 ±
19.35 ±
14.28 ±
11.17 ±
4.15 ±
0.30b
0.13c'd
0.74C'd
7.20
1.00C
0.66C
0.54
0.42
PbF-PbM
7.75
4.80
2.72
30.00
15.38
11.58
12.34
3.96
16
± 0.50C
± 0.19°
± 0.70C
± 8.20C
± 1.10C
± 0.78C
±1.24
± 0.46
,d,e
,d,e
,d,f
,d,f
,d,f
aCF = control female; CM = control male; PbM = lead-treated male; PbF = lead-treated female.
Significantly (p <0.05) less than mean for CF-CM.
Significantly (p <0.01) less than mean for CF-CM.
Significantly (p <0.01) less than mean for CF-PbM.
Significantly (p <0.01) less than mean for PbF-CM.
Significantly (p <0.05) less than mean for PbF-CM.
Source: Stowe and Goyer (1971).
-------
high dose. Jacquet and co-workers described effects of maternal dietary lead exposure on pre-
implantation mouse embryos (Jacquet, 1976; Jacquet et a!., 1976). They found lead in the diet
to be associated with retardation of cleavage in embryos, failure of trophoblastic giant cells
to differentiate, and absence of a uterine decidual reaction. Maisin et al. (1978) also found
delayed cleavage in embryos of mice fed lead acetate prior to mating and up to 7 days after-
wards.
Giavini et al. (1980) further confirmed the ability of lead to affect the preimplantation
embryo in studies of rats transplacentally exposed to lead nitrate,-and Wide and Nilsson
(1977, 1979) reported that inorganic lead had similar effects on mice. Jacquet (1978) was
able to force implantation in that species by use of high doses of progesterone, while Wide
(1980) determined that administration of estradiol-17p and progesterone could reverse the
effects of lead on implantation. Wide suggested that the lead-induced implantation blockage
was mediated by a decrease in endometrial responsiveness to both sex steroids. Jacquet (1976)
and Jacquet et al. (1977b) had attributed lead-induced prevention of implantation in the mouse
to a lack of endogenous progesterone alone, stating that estrogen levels were unaffected.
Later, however, Jacquet et al. (1977a) stated that estrogen levels also decreased, a finding
not supported by Wide and Wide (1980). The latter authors did find a lead-induced increase in
uterine estradiol receptors, but no change in binding affinities. Although sex steroids
appear to be involved in lead's effects on implantation in rodents, the precise mechanism is
not clear.
In order to examine lead's effects early in gestation, Wide and Nilsson (1977) examined
embryos from untreated mice and from mothers given 1 mg lead chloride on days 3, 4, or 6 of
pregnancy. Embryonic mortality was greater in lead-treated litters; in the day-6 group some
abnormal embryos were observed by day 8. In a later experiment, Wide (1978) removed blasto-
cysts from lead-treated mice. She found that they attached and grew normally during three
days of jn vitro culture. Other blastocysts from untreated mothers were cultured in media
containing lead, and a dose-dependent decrease in the number of normally developing embryos
was seen. Wide (1983) then transplanted blastocysts from mice treated with an implantation-
inhibiting lead dose and found that they implanted and developed normally in foster mothers.
In a more recent study, Molls et al. (1983) exposed two-cell mouse embryos to 0.1 or
1.0 ^g lead chloride per ml of culture medium. By 64 hours of incubation, both treatment
levels had resulted in decreased cell proliferation. Cell death was also seen in morula stage
lead-treated embryos. Exposure to x-rays one hour after the start of incubation had an addi-
tional (but not synergistic) effect.
A study employing domestic sheep was reported by Sharma and Buck (1976), who fed lead
powder to pregnant ewes throughout gestation. Levels in the diet were varied from 0.5 to 16
mg/kg per day in an effort to keep blood lead levels near 40 ug/dl (actual levels ranged from
12-213
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30 to 70 ug/dl). Such treatment resulted in a greatly decreased lambing percentage but no
gross malformations. However, only 11 lead-treated and 9 control subjects were studied.
12.6.2.2.4 Teratogenicity and prenatal toxicity of lead in animals.
12.6.2.2.4.1 High dose effects on the conceptus. Teratogenic effects refer to physical
defects (malformations) in the developing offspring. Prenatal toxicity (embryotoxicity, feto-
toxicity) includes premature birth, prenatal death, stunting, histopathological effects, and
transient biochemical or physiological changes. Behavioral teratogenicity, consisting of be-
havioral alterations or functional (e.g., motor, sensory) deficits resulting from in uterq
exposure, is considered in Section 12.4.3 of this chapter.
Teratogenicity of lead, at high exposure levels, has been demonstrated in rodents and
birds, with some results suggesting a species-related specificity of certain gross teratogenic
effects. Perm and Carpenter (1967), as well as Perm and Perm (1971), reported increased
embryonic resorption and malformation rates when various lead salts were administered i.v. to
pregnant hamsters. Teratogenic effects were largely restricted to the tail region, including
malformations of sacral and caudal vertebrae resulting in absent or stunted tails. Gale
(1978) found the same effects, plus hydrocephalus, among six strains of hamsters and noted
differences in susceptibility, suggesting a genetic component in lead-induced teratogenicity.
Zegarska et al. (1974) performed a study with rats injected with lead acetate at mid-ges-
tation. They reported embryonic mortality and malformations. McClain and Becker (1975) sub-
sequently administered lead nitrate i.v. to rats on one of days 8-17 of gestation, producing
malformations and embryo- and fetotoxicity. Hackett et al. (1978, 1982a,b) also gave lead
i.v. to rats and found malformations and high incidences of prenatal mortality. Minsker et
al. (1982) gave lead i.v. to dams on day 17 of gestation and observed decreased birth weights
as well as decreased weight and survival by postpartum day 7.
In another study, Miller et al. (1982) used oral doses of lead acetate up to 100 mg/kg
given daily to rats before breeding and throughout pregnancy and found fetal stunting at the
high dose, but no other effects. Maternal blood lead values ranged from 80 to 92 ug/dl prior
to mating and from 53 to 92 ug/dl during pregnancy. Pretreatment and control blood lead
levels averaged 6-10 ug/dl. Also, Warden et al. (1982) gavaged rats daily with lead doses of
up to 150 mg/kg from gestation day 6-18 and observed decreased prenatal survival at the high
dose, but no malformations.
Perm (1969) reported that teratogenic effects of i.v. lead in hamsters are potentiated in
the presence of cadmium, leading to severe caudal dysplasia. This finding was duplicated by
Hilbelink (1980). In addition to caudal malformations, lead appears to influence the morphol-
ogy of the developing brain. Por example, Murray et al. (1978) described a significant
decrease in number of dendritic spines and observed a variety of morphological abnormalities
of such spines in the parietal cortex of 30-day-old rat pups exposed to lead during gestation
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and nursing, during the postweam'ng period only, or during both periods. Morphometric analy-
sis of rats transplacentally exposed to lead indicated that cellular organelles were altered
as a function of dose and stage of development at exposure (Klein et al., 1978). These
results indicate that morphologically apparent effects of lead on the brain could be produced
by exposure during pregnancy alone, a question not addressed by Murray et al. (1978). See
Section 12.4.3 for a discussion of other studies relating lead exposure to morphological and
functional alterations in the CMS of developing animals.
12.6.2.2.4.2 Low-dose effects on the conceptus. There is a paucity of information re-
garding the teratogenicity and developmental toxicity of prolonged low-level lead exposure.
Kimmel et al. (1980) exposed female rats chronically to lead acetate via drinking water (0.5,
5, 50, and 250 ppm) from weaning through mating, gestation, and lactation. They observed a
decrease in fetal body length of female offspring at the high dose, and the female offspring
from the 50 and 250 ppm groups weighed less at weaning and showed delays in physical develop-
ment. Maternal toxicity was evident in the rats given 25 ppm or higher doses, corresponding
to blood lead levels of 20 ug/dl or higher. Reiter et al. (1975) observed delays in the
development of the nervous system in offspring exposed to 50 ppm lead throughout gestation and
lactation. Whether these delays in development resulted from a direct effect of lead on the
nervous system of the pups or reflect secondary changes (resulting from malnutrition, hormonal
imbalance, etc.) is not clear. Whatever the mechanisms involved, these studies suggest that
low-level, chronic exposure to lead may induce postnatal developmental delays.
12.6.2.2.4.3 Prenatal effects of organolead compounds. In an initial study of the ef-
fects of organolead compounds in animals, McClain and Becker (1972) treated rats orally with
7.5-30 mg/kg tetraethyl lead, 40-160 mg/kg tetramethyl lead, or 15-38 mg/kg trimethyl lead
chloride, given in three divided doses on gestation days 9-11 or 12-14. The last compound was
also given intravenously at doses of 20-40 mg/kg on one of days 8-15 of pregnancy. The
highest dose of each agent resulted in maternal death, while lower doses caused maternal toxi-
city. At all dose levels, fetuses from dams given multiple treatment weighed less than con-
trols. Single treatments at the highest doses tended to have similar effects. In some cases
delayed ossification was observed. In addition, direct intra-amniotic injection of trimethyl
lead chloride at levels up to 100 ug per fetus caused increasing fetal mortality.
Kennedy et al. (1975) administered tetraethyl lead by gavage to mice and rats during the
period of organogenesis at dose levels up to 10 mg/kg. Maternal toxicity, prenatal mortality,
and developmental retardation were noted at the highest doses in both species, although mater-
nal treatment was discontinued after only three days due to excessive toxicity. In a subse-
quent study involving alkyl lead, Odenbro and Kihlstrom (1977) treated female mice orally with
triethyl lead at doses of up to 3.0 rag/kg per day on days 3-5 following mating. The highest
treatment levels resulted in decreased pregnancy rates, while at 1.5 mg/kg, lower implantation
12-215
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rates were seen. In order to elucidate the mechanism of implantation failure in organolead-
intoxicated mice, Odenbro et al. (1982) measured plasma sex steroid levels in mice five days
after mating. Levels of both estradiol and progesterone, but not estrone, were decreased
following intraperitoneal triethyl lead chloride on days three and four of gestation. Such
results suggest a hormonal mechanism for blockage of implantation, a finding also suggested
for inorganic lead (Wide, 1980; Jacquet et al., 1977a). In an attempt to elucidate the
mechanism by which organolead compounds decrease fetal growth, Kihlstrom and Odenbro (1983)
treated guinea pigs with i.p. triethyl lead chloride. They observed reduced placental trans-
fer of alpha-amino isobutyrate following doses of 2.5 mg/kg, but no effect was seen at 1.0
rag/kg.
12.6.2.2.4.4 Effects of lead on fetal phy"^^Y and metabolism. Biochemical indicators
of developmental toxicity have been the subject of a number of investigations, as possible in-
dicators of subtle prenatal effects. Hubermont et al. (1976) exposed female rats to lead in
drinking water before mating, during pregnancy, and after delivery. In the highest exposure
group (10 ppm), maternal and offspring blood lead values were elevated and approached 68 and
42 ug/dl, respectively. Inhibition of ALA-D and elevation of free tissue porphyrins were also
noted in the newborns. Maternal diets containing up to 0.5 percent lead were associated with
increased fetal porphyrins and decreased ALA-D activity by Jacquet et al. (1977a). Fetuses in
the high-dose group had decreased weights, but no data were presented on maternal weight gain
or food consumption (which could have influenced fetal weight).
Fetal effects were also investigated by Hayashi (1983a,b), who reported that lead levels
as low as 5 ppm in the drinking water of rats for the first 18-21 days of pregnancy resulted
in decreased ALA-D activity in the fetal erythrocytes. Fetal hepatic ALA-D activity was
increased in the lead-treated groups, while hematocrit and hemoglobin concentrations were de-
creased by day 21. Fetal blood leads were 27 ± 16 and 19 ± 10 ug/dl in the 18- and 21-day
groups, respectively.
In the only inhalation exposure study (Prigge and Greve, 1977), rats were exposed
throughout gestation to an aerosol containing 1, 3, or 10 mg Pb/m3 or to a combination of 3 mg
Pb/m3 and 500 ppm carbon monoxide (CO). Both maternal and fetal ALA-D activities were strong-
ly inhibited by lead exposure in a dose-related manner. In the presence of lead plus CO, how-
ever, fetal (but not maternal) ALA-D activity was higher than in the group given lead alone,
possibly due to the increase in total ALA-D seen in the CO-plus-lead treated fetuses. Fetal
body weight and hematocrit were decreased in the high-dose lead group, while maternal values
were unchanged, thus suggesting that the fetuses were more sensitive to lead's effects than
were the mothers. Granahan and Huber (1978) also reported decreased hematocrit, as well as
reduced hemoglobin levels, in fetal rats from lead-intoxicated dams (1000 ppm in the diet
throughout gestation).
12-216
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Gerber and Maes (1978) fed pregnant mice diets containing up to one percent lead from day
7 to 18 of pregnancy and determined levels of heme synthesis. Incorporation of iron into
fetal heme was inhibited, but glycine incorporation into heme and protein was unaffected.
Gerber et al. (1978) also found that dietary lead given late in gestation resulted in dimin-
ished placental blood flow but did not decrease uptake of a non-metabolizable amino acid,
alpha-ami no isobutyrate. The authors could not determine whether lead-induced fetal growth
retardation was due to placental insufficiency or to the previously described reduction in
heme synthesis (Gerber and Maes, 1978). They did not mention the possibility that the treated
mothers may have reduced their food consumption, resulting in a reduced nutrient supply to the
fetus, regardless of fetal ability to absorb nutrients.
In another study where evidence of physiological changes was seen at low lead levels,
rats were given 0, 5, 25, 100, or 500 ppm lead in their drinking water throughout gestation
and lactation (Victery et al., 1983). Their offspring were tested at one month of age and
plasma renin activity was found to be elevated at all dose levels, while renal renin concen-
trations were elevated at the two highest doses. The increases in plasma angiotensin II
(All) levels found in the offspring of rats treated with 100 and 500 ppm lead were partially
inhibited when the one-month-old pups were anesthetized and subjected to a surgical procedure
(laparotomy) prior to sampling. Such results suggest that exposure to relatively low lead
levels during development and via nursing may enhance basal renin secretion in young rats,
while at least at higher levels (the two low-dose groups were not tested for All), such treat-
ment tended to inhibit the response to renin-releasing conditions.
More recently, Wardell et al. (1982) exposed rat fetuses 21} utero to lead by gavaging
their pregnant mothers with 150 mg/kg on gestation days 6-18. On day 19, fetal limb
cartilage was tested for ability to synthesize protein, DNA, and proteoglycans, but no adverse
effects were seen. Also, Talcott and Koller (1983) found no effect on the immune system of
the offspring of mice exposed during gestation to 1000 ppm dietary lead with or without
Aroclor 1254.
12.6.2.2.4.5 Possible mechanisms of lead-induced teratogenesis. The reasons for the
localization of many of the gross teratogenic effects of lead are unknown at this time. Perm
and Perm (1971) have suggested that the observed specificity could be explained by an inter-
ference with specific enzymatic events. Lead alters mitochondrial function and enhances or
inhibits enzymes (see Section 12.2.1); any or all such effects could ultimately interfere with
normal development. Similarly, inhibition of ALA production has been suggested as a mechanism
of teratogenesis by Cole and Cole (1976), while Danielsson et al. (1983) have proposed that
lead's teratogenic effects may be based in part on a functional oxygen deficiency in certain
tissues due to an interference with fetal heme production.
12-217
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In an attempt to study the mechanics of lead induction of sacral-tail region malforma-
tions, Carpenter and Perm (1977) examined hamster embryos treated at mid-gestation during the
critical stage for response to teratogens in this species. The initial effects were edema of
the tail region of embryos 30 hours after maternal exposure, followed by blisters and hema-
tomas. These events disrupted normal caudal development, presumably by mechanical displace-
ment. The end results seen in surviving fetuses were missing, stunted, or malformed tails and
anomalies of the lower spinal cord and adjacent vertebrae.
12.6.2.2.4.6 Maternal factors in lead-induced teratogenesis and fetotoxicity. Nutri-
tional factors may also have a bearing on the prenatal toxicity of lead. Jacquet and Gerber
(1979) reported increased mortality and defects in fetuses of mice given intraperitoneal
injections of lead while consuming a calcium-deficient diet during gestation. In several
treatment groups, lead-treated calcium-deficient mothers had low blood calcium levels, while
controls on the same diet had normal values. It is not certain how meaningful these data are,
however, as there was no clear dose-response relationship within diet groups. In fact, fetal
weights were said to be significantly higher in two of the lead-treated groups (on the normal
diet) than in the untreated controls. Another problem with the study was that litter numbers
were smal1.
In a later study, Carpenter (1982) reported greater prenatal mortality and incidence of
malformations in fetal hamsters from mothers given 0.05 or 0.1 percent lead acetate in their
drinking water if the mothers were also on diets deficient in either calcium or iron. Numbers
of litters per group were small, however, and the two lead dose groups were combined when the
data were averaged, making the results difficult to interpret.
Another study on interactions of lead with other elements was done by Dilts and Ahokas
(1979), who exposed rats to lead in their drinking water throughout gestation. Controls were
pair-fed or fed ad libitum. Lead treatment was said to result in decreased fetal weight, and
dietary zinc supplementation was claimed to be associated with a protective effect against
fetal stunting. The data as presented do not allow the differentiation of effects due to
maternal stress (e.g., decreased food consumption) from direct effects on the fetus. In addi-
tion, litter numbers were small, and some of the data were confusing. For example, a lead-
treated and a pair-fed group had very similar litter sizes and total litter weights, but
rather dissimilar average fetal weights; also, dividing live litter weight by live litter size
does not give the authors' values for average fetal weight. Finally, no data were given on
maternal or fetal lead or zinc levels. In a further report on apparently the same animals as
above, Dilts and Ahokas (1980) found that lead inhibited cell division and decreased protein
contents of the fetal placentas, eviscerated carcasses, and livers. Such lead-related effects
were not influenced by maternal zinc supplementation.
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12.6.2.3 Effects of Lead on Avian Species. The effects of lead on the reproduction and
development of various avian species have been studied by a number of investigators, primarily
because of interest in the effects of lead shot ingested by wildlife or in order to develop an
avian embryo model for the experimental analysis of ontogenetic processes. The relevance of
such studies to the health effects of lead on humans is not clear. Consequently, these
studies are not discussed further here.
12.6.3 Summary
The most clear-cut data described in this section on reproduction and development are de-
rived from studies employing high lead doses in laboratory animals. There is still a need for
more critical research to evaluate the possible subtle toxic effects of lead on the fetus,
using biochemical, ultrastructural, or behavioral endpoints. An exhaustive evaluation of
lead-associated changes in offspring should include consideration of possible effects due to
paternal lead burden as well. Neonatal lead intake via consumption of milk from lead-exposed
mothers may also be a factor at times. Moreover, it must be recognized that lead's effects on
reproduction may be exacerbated by other environmental factors (e.g., dietary influences,
maternal hyperthermia, hypoxia, and co-exposure to other toxins).
There are currently no reliable data pointing to adverse effects in human offspring fol-
lowing lead exposure of fathers per se. Early studies of pregnant women exposed to high
levels of lead indicated toxic, but not teratogenic, effects on the conceptus. Unfortunately,
the collective human data regarding lead's effects on reproduction or HI utero development
currently do not lend themselves to accurate estimation of exposure-effect or no-effect
levels. This is particularly true regarding lead effects on reproductive performance in
women, which have not been well documented at low exposure levels. Still, prudence would
argue for avoidance of lead exposures resulting in blood lead levels exceeding 25-30 [iq/dl in
pregnant women or women of child-bearing age in general, given the equilibration between
maternal and fetal blood lead concentrations that occurs and the growing evidence for dele-
terious effects in young children as blood lead levels approach or exceed 25-30 ug/dl. Indus-
trial exposure of men to lead at levels resulting in blood lead values of 40-50 ug/dl also
appear to result in altered testicular function.
The paucity of human exposure data forces an examination of the animal studies for indi-
cations of threshold levels for effects of lead on the conceptus. It must be noted that the
animal data are almost entirely derived from rodents. Based on these rodent data, it seems
likely that fetotoxic effects have occurred in animals at chronic exposures to 600-800 ppm
inorganic lead in the diet. Subtle effects appear to have been observed at 5-10 ppm in the
drinking water, while effects of inhaled lead have been seen at levels of 10 mg/m3. With
multiple exposure by gavage, the lowest observed effect level is 64 mg/kg per day, and for
12-219
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exposure via injection, acute doses of 10-16 mg/kg appear effective. Since humans are most
likely to be exposed to lead in their diet, air, or water, the data from other routes of expo-
sure are of less value in estimating harmful exposures. Indeed, it appears that teratogenic
effects occur in experimental animals only when the maternal dose is given by injection.
Although human and animal responses may be dissimilar, the animal evidence does document
a variety of effects of lead exposure on reproduction and development. Measured or apparent
changes in production of or response to reproductive hormones, toxic effects on the gonads,
and toxic or teratogenic effects on the conceptus have all been reported. The animal data
also suggest subtle effects on such parameters as metabolism and cell structure that should be
monitored in human populations. Well-designed human epidemiological studies involving large
numbers of subjects are still needed. Such data could clarify the relationship of exposure
levels and durations to blood lead values associated with significant effects and are needed
for estimation of no-effect levels. (Recent studies, most of which are prospective epidemiol-
ogical investigations, on the relationship between relatively low-level lead exposure and
effects on fetal and child development, along with supporting experimental evidence on
possible underlying mechanisms, are reviewed in an Addendum to this document.)
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12.7 GENOTOXIC AND CARCINOGENIC EFFECTS OF LEAD
12.7.1 Introduction
Potential carcinogenic, genotoxic (referring to alteration in structure or metabolism of
DMA), and mutagenic roles of lead are considered here. Epidemiological studies of occupation-
ally exposed populations are considered first. Such studies investigate possible associations
of lead with induction of human neoplasia and are important because they assess the incidence
of disease in humans under actual ambient exposure conditions. However, such studies have
many limitations that make it difficult to assess the carcinogenic activity of any specific
agent. These include general problems in accurately determining the amount and nature of
exposure to a particular chemical agent; in the absence of adequate exposure data it is diffi-
cult to determine whether each individual in a population was equally exposed to the agent in
question. It is also often difficult to assess other factors, such as exposure to carcinogens
in the diet, and to control for confounding variables that may have contributed to the inci-
dence of any neoplasms. These factors tend to obscure the effect of lead alone. Also, in an
occupational setting a worker is often exposed to various chemical compounds, making it more
difficult to assess epidemiologically the injurious effect resulting specifically from expo-
sure to one, such as lead.
A second approach considered here examines the ability of specific lead compounds to in-
duce tumors in experimental animals. The advantage of these studies over epidemiological in-
vestigations is that a specific lead compound, its mode of administration, and level of expo-
sure can be well defined and controlled. Additionally, many experimental procedures can be
performed on animals that for ethical reasons cannot be performed on humans, thereby allowing
a better understanding of the course of chemically induced injury. For example, animals may
be sacrificed and necropsies performed at any desired time during the study. Factors such as
diet and exposure to other environmental conditions can be well controlled, and genetic vari-
ability can be minimized by use of well established and characterized animal lines. One
problem with animal studies is the difficulty of extrapolating such data to humans. However,
this drawback is perhaps more important in assessing the toxicity of organic chemicals than in
assessing inorganic agents, because the injury induced by many organic agents is highly depen-
dent upon reactive intermediates formed ;ni vivo by enzymatic action (e.g., microsomal enzymes)
upon the parent compound. In addition, both qualitative and quantitative differences between
the metabolic capabilities of humans and experimental animals have been documented (Neal,
1980). With inorganic compounds of lead, however, the element of interest undergoes little
alteration jn vivo and, therefore, the ultimate toxic agent is less likely to differ between
experimental animals and humans (Costa, 1980). The carcinogenic action of most organic chemi-
cals is dependent upon activation of a parent pro-carcinogen, whereas most metallic carcino-
gens undergo little alteration HI vivo to produce their oncogenic effects (Costa, 1980).
12-221
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A third approach discussed below is in vitro studies. Animal carcinogen bioassays are
currently the preferred means for assessing carcinogenic activity but they are extremely ex-
pensive and time consuming. As a result, much effort has been directed toward developing
suitable iji vitro tests to complement iji vivo animal studies for the evaluation of the poten-
tial oncogenicity of chemicals. The cell transformation assay has as its endpoint neoplastic
transformation of mammalian cells and is the most suitable jn vitro system because it examines
cellular events closely related to carcinogenesis (Heck and Costa, 1982a). A general problem
with this assay system, which is less troublesome with reference to metal compounds, is that
it employs fibroblastic cells in culture, which lack many jji vivo metabolic systems. Since
lead is not extensively metabolized jn vivo, addition of liver microsomal extracts (which has
been attempted in this and similar systems) is not necessary to generate the ultimate carcino-
gen^) from this metal (see above). However, if other indirect factors are involved with lead
carcinogenesis in vivo, then these might be absent in such culture systems (e.g., specific
lead-binding proteins that direct lead interactions JH vivo with oncogenically relevant
sites). There are also technical problems related to the culturing of primary cells and dif-
ficulties with the final microscopic evaluation of morphological transformations, which are
prone to some subjectivity. However, if the assay is performed properly it can be very relia-
ble and reproducible. Modifications of this assay system (i.e., exposure of pregnant hamsters
to a test chemical followed by culturing and examination of embryonic cells for transplacen-
tally induced transformation) are available for evaluation of iji vivo metabolic influences,
provided that the test agent is transported to the fetus. Additionally, cryopreservation of
primary cultures isolated from the same litter of embryos can control for variation in cell
populations exposed to test chemicals and give more reproducible responses in replicate ex-
periments (Pienta, 1980). A potential advantage of the cell transformation assay system is
the possibility that cultured human cells can be transformed jn vitro. Despite numerous at-
tempts, however, no reproducible human-cell transformation system has yet been sucessfully
established which has been evaluated with a number of different chemicals of defined carcino-
genic activity.
Numerous processes have been closely linked with oncogenic development, and specific
assay systems that utilize events linked mechanistically with cancer as an endpoint have been
developed to probe whether a chemical agent can affect any of these events. These systems in-
clude assays for mutations, chromosomal aberrations, development of micronuclei, enhancement
of sister chromatid exchange, effects on DNA structure, and effects on DNA and RNA polymerase.
These assay systems have been used to examine the genotoxicity of lead and facilitate the
assessment of possible lead carcinogenicity. Chromosomal aberration studies are useful
because human lymphocytes cultured from individuals after exposure to lead allow evaluation of
12-222
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genotoxic activity that occurred under the influence of an ijn vivo metabolic system. Such
studies are discussed below in relationship to genotoxic effects of lead. However, a neoplas-
tic change does not necessarily result, and evaluations of some less conspicuous types of
chromosomal aberrations are somewhat subjective since microscopy is exclusively utilized in
the final analyses. Nevertheless, it is reasonable to assume that if an agent produces chro-
mosomal aberrations it may have potential carcinogenic activity. Many carcinogens are also
mutagenic, and this fact, combined with the low cost and ease with which bacterial mutation
assays can be performed, has resulted in wide use of these systems in determining potential
careinogenicity of chemicals. Mutation assays can also be performed with eukaryotic cells and
several studies are discussed below that examined the mutagenic role of lead in these systems.
However, in bacterial systems such as the Ames test, metal compounds with known human carcino-
genic activity are generally negative and, therefore, this system is not useful for determin-
ing the potential oncogenicity of lead. Similarly, even in eukaryotic systems, metals with
known human cancer-causing activity do not produce consistent mutagenic responses. Reasons
for this lack of mutagenic effect remain unclear, and it appears that mutagenicity studies of
lead cannot be weighed heavily in assessing its genotoxicity.
Other test systems that probe for effects of chemical agents on DMA structure may be use-
ful in assessing the genotoxic potential of lead. Sister chromatid exchange represents the
normal movement of DNA in the genome and enhancement of this process by potentially carcino-
genic agents is a sensitive indicator of genotoxicity (Sandberg, 1982). Numerous recently
developed techniques can be used to assess DNA damage induced by chemical carcinogens. One of
the most sensitive is alkaline elution (Kohn et al., 1981), which may be used to study DNA
lesions produced jn vivo or in cell culture. This technique can measure DNA strand breaks or
crosslinks in DNA, as well as repair of these lesions, but the toxicity of lead compounds has
not been studied with this technique. Assessment of the induction of DNA repair represents
one of the most sensitive techniques for probing genotoxic effects. The reason for this is
that the other procedures measure DNA lesions that have persisted either because they were not
recognized by repair enzymes or because their number was sufficiently great to saturate DNA
repair systems. Measurement of DNA repair activation is still possible even if the DNA lesion
has been repaired, but effects of lead compounds on DNA repair have not been studied. There
are a few isolated experiments within publications that examined the ability of lead compounds
to induce DNA damage, but this line of investigation requires further work. There are some
well-conducted jn vitro studies of the effect of lead along with other water soluble metals on
isolated DNA and RNA polymerases, which suggest mutagenic mechanisms occurring in intact
cells. The ability of lead to affect the transcription of DNA and RNA merits concern in
regard to its potential oncogenic and mutagenic properties.
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12.7.2 Carcinogenesis Studies of Lead and its Compounds
12.7.2.1 Human Epidemiclogical Studies. Epidemiologies! studies of industrial workers, where
the potential for lead exposure is usually greater than for a "normal population," have been
conducted to evaluate the role of lead in the induction of human neoplasia (Cooper, 1976,
1981; Cooper and Gaffey, 1975; Chrusciel, 1975; Dingwall-Fordyce and Lane, 1963; Lane, 1964;
McMichael and Johnson, 1982; Neal et al., 1941; Nelson et al., 1982). In general, these
studies made no attempt to consider types of lead compounds to which workers were exposed or
to determine probable routes of exposure. Some information on specific lead compounds encoun-
tered in the various occupational settings, along with probable exposure routes, would have
made the studies more interpretable and useful. As noted in Chapter 3, with the exception of
lead nitrate and lead acetate, many inorganic lead salts are relatively water insoluble. If
exposure occurred by ingestion, the ability of water-insoluble lead salts (e.g., lead oxide
and lead sulfide) to dissolve in the gastrointestinal tract may contribute to understanding of
their ultimate systemic effects in comparison to their local actions in the gastrointestinal
tract. Factors such as particle size are also important in the dissolution of any water in-
soluble compounds in the gastrointestinal system (Mahaffey, 1983). When considering other
routes of exposure (e.g., inhalation), the water solubility of the lead compound in question,
as well as the particle size, are extremely important, both in terms of systemic absorption
and contained injury in the immediate locus of the retained particle (see Chapter 10). A
hypothetical example is the inhalation of an aerosol of lead oxide versus a water soluble lead
salt such as lead acetate. Lead oxide particles having a diameter of <5 Mm would tend to de-
posit in the lung and remain in contact with cells there until they dissolved, while soluble
lead salts would dissipate systemically at a much more rapid rate. Therefore, in the case of
inhaled particulate compounds, localized exposure to lead might produce injury primarily in
respiratory tissue, whereas with soluble salts, systemic (i.e., CNS, kidney, and erythropoie-
tic) effects might predominate.
The studies of Cooper and Gaffey (1975) and Cooper (1976, 1981) examined the incidence of
cancer in a large population of industrial workers exposed to lead. Two groups of individuals
were identified as the lead-exposed population under consideration: smelter workers from six
lead production facilities and battery plant workers (Cooper and Gaffey, 1975). The authors
reported (see Table 12-17) that total mortality from cancer was higher in lead smelter workers
than in a control population in two ways: (1) the difference between observed and expected
values for the types of malignancies reported; and (2) the standardized mortality ratio (SMR),
which, by comparison to a control population, indicates a greater than "normal" (but not nec-
essarily statistically significant) response if it is in excess of 100 percent. These studies
report not only an excess of all forms of cancer in smelter workers but also a greater level
of cancer in the respiratory and digestive systems in both battery plant and smelter workers.
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TABLE 12-17. EXPECTED AND OBSERVED DEATHS AND STANDARDIZED MORTALITY RATIOS FOR MALIGNANT
NEOPLASMS FROM JAN. 1, 1947 TO DEC. 31, 1979 FOR LEAD SMELTER AND BATTERY PLANT WORKERS
Causes.of death
(ICDa Code)
All malignant neoplasms (140-205)
Buccal cavity & pharynx (140-148)
Digestive organs peritoneum (150-159)
Respiratory system (160-164)
Genital organs (170-179)
Urinary organs (180-181)
Leukemia (204)
Lymphosarcoma, lymphatic, and
hematopoietic (200-203, 205)
Other sites
Smelters
Observed
69
0
25
22
4
5
2
3
8
Expected
54.95
1.89
17.63
15.76
4.15
2.95
2.40
3.46
6.71
h
5MRD
133
—
150
148
101
179
88
92
126
Battery Plant
Observed
186
6
70
61
8
5
6
7
23
Expected
180.34
6.02
61.48
49.51
18.57
10.33
7.30
9.74
17.39
5MIT
111
107
123
132
46
52
88
77
142
International Classification of Diseases.
Correction of +5.55% applied for 18 missing death certificates; SMR = standardized mortality
ratio.
Correction of +7.52% applied for 71 missing death certificates.
Source: Cooper and Gaffey (1975).
The incidence of urinary system cancer was also elevated in the smelter workers (but not in
the battery plant workers), although the number of individuals who died from this neoplasm was
very small. As the table indicates, death from neoplasm at other sites was also elevated com-
pared with a normal population, but these results were not discussed in Cooper and Gaffey1s
(1975) report, since these elevated incidences of cancer were not statistically significant by
their analysis.
Kang et al. (1980) examined the Cooper and Gaffey (1975) report and noted an error in the
statistical equation used to assess the significance of excess cancer mortality. Table 12-18,
from Kang et al. (1980) shows results based on what they claimed was a corrected form of the
statistical equation previously used by Cooper and Gaffey (1975); it also employed another
statistical test claimed to be more appropriate. Statistical significance was observed in
every category listed with the exception of battery plant workers, whose deaths from all forms
of neoplasia were not different from a control population. Gaffey (1980), in responding to
the letter of Kang et al. (1980), indicated that a typographical error had been made in the
equation printed in their publication (Cooper and Gaffey, 1975) but that the correct equation
had actually been used in assessing the statistical significance of their data.
Cooper and Gaffey (1975) did not discuss types of lead compounds that these workers may
have been exposed to in smelting operations, but workers thus employed likely ingested or
12-225
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TABLE 12-18. EXPECTED AND OBSERVED DEATHS RESULTING FROM SPECIFIED MALIGNANT NEOPLASMS
FOR LEAD SMELTER AND BATTERY PLANT WORKERS AND LEVELS OF SIGNIFICANCE BY
TYPE OF STATISTICAL ANALYSIS ACCORDING TO ONE-TAILED TESTS
Probability
Number of deaths
Causes of death
(ICDa code)
Ob-
served
Ex- ,
pected SMRD
Pois-
son
This
anal ^
ysis
Cooper
and
Gaffey6
Lead smelter workers:
69
25
All malignant neoplasms
(140-205)
Cancer of the digestive organs,
peritoneum (250-159)
Cancer of the respiratory system 22
(160-164)
Battery plant workers:
54.95 133 <0.02 <0.01 <0.02
17.63 150 <0.03 <0.02 <0.05
15.76 148 <0.05 <0.03 >0.05
All malignant neoplasms
(140-205)
Cancer of the digestive organs,
peritoneum (150-159)
Cancer of the respiratory system
(160-164)
186
70
61
180.34
61.48
49.51
111
123
132
>0.05
<0.05
<0.03
>0.05
<0.04
<0.02
>0.05
>0.05
<0.03
International Classification of Diseases.
Standardized mortality ratios (SMRs) were corrected by Cooper and Gaffey for missing death
certificates under the assumption that distribution of causes of death was the same in
missing certificates as in those that were obtained.
Observed deaths were recalculated as follows: adjusted observed deaths = (given SMR/100) x
expected deaths.
JGiven 2 = (SMR - 100) ,/expected/lOO.
eGiven 2 = (SMR - 100)//100 x SMR/expected.
Source: Kang et al. (1980).
inhaled oxides and sulfides of lead. Since these and other lead compounds produced in the in-
dustrial setting are not readily soluble in water it could be that the cancers arising in res-
piratory or gastrointestinal systems were caused by exposure to water-insoluble lead com-
pounds. Although the Cooper and Gaffey (1975) study had a large sample (7032), only 2275 of
the workers (32.4 percent) were employed when plants monitored urinary lead. Urinary lead
values were available for only 9.7 percent of the 1356 deceased employees on whom the cancer
mortality data were based. Only 23 (2 percent) of the 1356 decedents had blood lead levels
measured. Cooper and Gaffey (1975) did report some average urinary and blood lead levels,
12-226
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where 10 or more urine or at least three blood samples were taken (viz., battery plant
workers: urine lead = 129 ug/1, blood lead = 67 ug/dl; smelter workers: urine lead = 73
(jg/1, blood lead = 79.7 ug/dl). Cooper (1976) noted that these workers were potentially
exposed to other materials, including arsenic, cadmium, and sulfur dioxide, although no data
on such exposures were reported. In these and other epidemiological studies in which selec-
tion of subjects for monitoring exposure to an agent such as lead is left to company discre-
tion, it is possible that individual subjects are monitored primarily on the basis of obvious
signs of lead exposure, while other individuals who show no symptoms of lead poisoning would
not be monitored (Cooper and Gaffey, 1975). It is also not clear from these studies when the
lead levels were measured, although the timing of measurement would make little difference
since no attempt was made to match an individual's lead exposure to any disease process.
In a follow-up study of the same population of workers, Cooper (1981) concluded that lead
had no significant role in the induction of neoplasia. However, he did report SMRs of 149
percent and 125 percent for all types of malignant neoplasms in lead battery plant workers
with <10 or >10 years of employment, respectively (Cooper, 1981). In battery workers employed
for 10 years or more there was an unusually high incidence of cancer listed as "other site"
tumors (SMR = 229 percent; expected = 4.85, observed = 16) (Cooper, 1981, Table 13). Respira-
tory cancers were elevated in the battery plant workers employed for less than 10 years
(SMR = 172 percent). Similarly, in workers involved with lead production facilities for more
than 10 years the SMR was 151 percent.
An analysis of data for a more carefully selected subset of the same population (6819
workers versus 7032 originally) for the period 1947-1980 was recently reported by Cooper
(1985). Deaths due to malignant neoplasms were elevated in both cohorts of workers (SMRs =
113 percent), a significant excess in battery workers but not in smelter workers because of a
smaller number of cases. Most of these deaths occurred prior to 1971, which accounts for the
lack of such findings in Cooper's (1981) analysis of 1971-1975 data. Consistent with earlier
findings, the primary tumor sites were the gastrointestinal tract and the lung. Cooper (1981,
1985) noted that the lack of information on smoking histories made interpretation of the res-
piratory cancers problematic. However, the association of lead exposure with gastric cancer
is consistent with findings of Sheffet et al. (1982), who reported an increased, albeit non-
significant, incidence of stomach cancer in workers exposed to lead chromate. Cooper (1985)
suggested that high local concentrations of ingested lead could have a co-carcinogenic effect,
particularly in those whose dietary or alcohol intake patterns predispose them to higher-than-
average gastric cancer rates. As noted by Cooper, further study of the possible association
between lead and gastric cancer seems advisable. At present, however, without better docu-
mentation of lead exposure histories, it is difficult to assess the degree of lead's contribu-
tion to the above findings.
12-227
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A recent study (McMichael and Johnson, 1982) examined the historical incidence of cancers
in a population of smelter workers diagnosed as having lead poisoning. The incidence of can-
cer in a relatively small group of 241 workers was compared with 695 deceased employees from
the same company. The control group had been employed during approximately the same period
and was asserted to be free from lead exposure, although there were no data to indicate lead
levels in either the control or the experimental group. Based upon diagnoses of lead poison-
ing made in the 1920s and 1930s for a majority of the deaths, the authors concluded that there
was a considerably lower incidence of cancer in lead-poisoned workers (McMichael and Johnson,
1982). However, there is no indication of how lead poisoning was diagnosed. It is difficult
to draw any conclusions from this study with regard to the role of lead in human neoplasia.
Davies (1984) found that workers exposed to both lead and zinc chromate in English pig-
ment factories showed a significantly increased incidence of lung cancer mortality after at
least one year of medium or high exposure. However, lung cancer mortality was normal in
workers exposed only to lead chromate, thus suggesting that zinc rather than lead chromate,
was the more significant risk factor.
Another recent epidemiological study (Selevan et al., 1984) has noted increased mortality
from renal cancer in a group of lead smelter workers. The SMR for deaths from renal cancer
was 204 percent for the entire cohort, although this excess mortality was not statistically
significant and only, represented 6 cases. However, of interest is the fact that the renal
cancers observed in humans in this study matched the types of cancers induced in experimental
animals by lead. This study also analyzed the number of deaths associated with high lead
exposure in combination with other contaminants (i.e., cadmium, zinc, and arsenic) as well as
those deaths associated predominantly with high lead exposure alone. The SMR for deaths from
renal cancer in the high lead exposure areas only was 301 percent. Similarly, deaths from
cancers of the urinary organs had an SMR of 199 percent in the high-lead-only group. These
results suggest that lead exposure could be associated with an increased incidence of renal
cancer in humans, but in the absence of statistical significance and corroboration by other
epidemiological studies, this finding should be interpreted with caution.
Two case studies have also suggested an association of lead exposure with renal cancer
(Baker et al., 1980; Lilis, 1981). The relatively high degree of lead exposure in these two
case reports was well documented by symptoms of lead intoxication and by measurements of blood
lead and erythrocyte protoporphyrin. Furthermore, Baker et al. (1980) found a relatively high
concentration of lead (~2.5 M9/9) in the patient's tumor as well as certain histologic simi-
larities to lead-induced neoplasms in animal kidneys (e.g., swollen mitochondria, numerous
dense lysosomes, and some amphophilic intranuclear inclusion bodies in epithelial cells adja-
cent to the proximal convoluted tubules). The generally sparse presence of intranuclear in-
clusion bodies in the patient's kidney was attributed to his use of oral penicillamine three
to four weeks prior to being examined.
12-228
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Conclusions regarding the ability of lead to induce human neoplasia must await further
epidemiological studies in which other factors that may contribute to the observed effects are
well controlled for and the disease process is assessed in individuals with well documented
exposure histories. Little can now be reliably concluded from available epidemiological
studies.
12.7.2.2 Induction of Tumors in Experimental Animals. As discussed in the preceding
sections, it is difficult to obtain conclusive evidence of the carcinogenic potential of an
agent using only epidemiological studies. Experiments testing the ability of lead to cause
cancer in experimental animals are an essential aspect of understanding its oncogenicity in
humans. However, a proper lifetime animal feeding study to assess the carcinogenic potential
of lead following National Cancer Institute guidelines (Sontag et al., 1976) has not been con-
ducted. The cost of such studies exceeds $1 million; consequently they are limited ohly to
those agents in which sufficient evidence based upon jr\ vitro or epidemiological studies
warrants such an undertaking. The literature on lead carcinogenesis contains many smaller
studies where only one or two doses were employed and where toxicological monitoring of ex-
perimental animals exposed to lead was generally absent. Some of these studies are summarized
in Table 12-19 (see also Section 12.8.2.2). Most mainly serve to illustrate that numerous
different laboratories have induced renal tumors in rats by feeding them diets containing 0.1
or 1.0 percent lead acetate. In some cases, other lead formulations were tested, but the
dosage selection was not based upon lethal dose values. In most cases, only one dose level
was used. Another problem with many of these studies was that the actual concentrations of
lead administered and internal body burdens achieved were not measured. Some of these studies
are discussed very briefly; others are omitted entirely because they contribute little to our
understanding of lead carcinogenesis.
Administration of 1.0 percent lead acetate (10,000 ppm) resulted in kidney damage and a
high incidence of mortality in most of the studies in Table 12-19. However, kidney tumors
were also evident at lower dosages (e.g., 0.1 percent lead acetate in the diet), which pro-
duced less mortality among the test animals. As discussed in Section 12.6, renal damage is
one of the primary toxic effects of lead. At 0.1 percent lead acetate (1000 ppm) in the diet,
the concentration of lead measured in the kidney was 30 |jg/g while 1 percent lead acetate re-
sulted in 300 ug/g of lead in the kidneys of necropsied animals (Azar et al., 1973). In most
of the studies with rats fed 0.1 or 1.0 percent lead in the diet, the incidence of kidney tu-
mors increased between the lower and higher dosage, suggesting a relationship between the de-
position of lead in the kidney and the carcinogenic response. Renal tumors were also induced
in mice at the 0.1 percent oral dosage of lead subacetate but not in hamsters that were simi-
larly exposed to this agent (Table 12-19).
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TABLE 12-19. EXAMPLES OF STUDIES ON THE INCIDENCE OF TUMORS IN EXPERIMENTAL
ANIMALS EXPOSED TO LEAD COMPOUNDS
Species
Rat
Rat
Rat
Mouse
Rat
Rat
Rat
Mouse
Rat
Hamster
Mouse
Rat
Rat
Pb compound
Pb phosphate
Pb acetate
Pb
subacetate
Pb
naphthenate
Pb phosphate
Pb
subacetate
Pb
subacetate
Tetraethyl
lead in
tricaprylin
Pb acetate
Pb
subacetate
Pb
subacetate
Pb nitrate
Pb acetate
Dose and mode*
120-680 rag
(total dose s.c.)
1% (in diet)
0.1% and
1.0% (in diet)
20% in benzene
(dermal 1-2
times weekly)
1.3 g (total
dosage s.c.)
0.5 - 1%
(in diet)
1% (in diet)
0.6 mg (s.c. )
4 doses between
birth and 21 days
3 ing/day for
2 months;
4 mg/day for
16 months (p.o. )
1.0% (in
0.5% diet)
0.1% and
1.0% (in diet)
25 g/1 (in
drinking water)
3 mg/day (p.o.)
Incidence (and type) of
neoplasms
19/29 (renal tumors)
15/16 (kidney tumors)
14/16 (renal carcinomas)
11/32 (renal tumors)
13/24 (renal tumors)
5/59 (renal neoplasms)
(no control with
benzene)
29/80 (renal tumors)
14/24 (renal tumors)
31/40 (renal tumors)
5/41 (lymphomas)
in females, 1/26 in
males, and 1/39 in
controls
72/126 (renal tumors)
23/94 males (testicular
[Leydig cell] tumors)
No significant incidence
of renal neoplasms
7/25 (renal carcinomas)
at 0.1%; substantial
death at 1.0%
No significant incidence
of tumors
89/94 (renal, pituitary,
cerebral gliomas,
adrenal, thyroid, pro-
Reference
Zol linger
(1953)
Boy land et
al. (1962)
Van Esch
et al. (1962)
Baldwin et
al. (1964)
Balo et al.
(1965)
Hass et al.
(1967)
Mao and
Molnar (1967)
Epstein and
Mantel (1968)
Zawirska and
Medras (1968)
Van Esch and
Kroes (1969)
Van Esch and
Kroes (1969)
Schroeder et
al. (1970)
Zawirska
and
Medras*. 1972
static, mammary tumors)
12-230
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TABLE 12-19. (continued)
Species Pb Compound Dose and mode
Incidence (and type) of
neoplasms
Reference
Rat
Pb acetate
Hamster Pb oxide
Rat
Pb chromate
0, 10, 50, 100,
1000, 2000 ppm
(in diet) for
2 yr
1 mg (intratracheal)
10 times
8 mg (i.m.)
for 9 monthly
injections
No tumors 0-100 ppm; Azar et al.
5/50 (renal tumors) at (1973)
500 ppm; 10/20 at 1000 ppm;
16/20 males, 7/20 females
at 2000 ppm
0/30 without benzopyrene,
12/30 with benzopyrene
(lung cancers)
Females: 2/25 lymphoma,
11/25 fibrosarcoma,
10/25 rhabdomyosarcoma,
1/25 osteogenic sarcoma
Males: 3/25 fibrosarcoma,
7/25 rhabdomyosarcoma,
3/25 renal carcinoma
Kobayashi
and
Okamoto (1974)
Furst et al.
(1976)
Mouse
Rat
Pb chromate 3 mg (i.m.)
for 4 monthly
injections
Pb acetate 0, 26, 2600 ppm
(in drinking water)
for 76 wk
Females only:
2/25 lymphoma,
3/25 lung carcinoma
81% (renal tumors)
at 2600 ppm
Furst et al.
(1976)
Koller et al.
(1985)
*s.c. = subcutaneous injection; p.o. = per os (gavage); i.m. = intramuscular injection.
Other lead compounds have also been tested in experimental animals, but in these studies
only one or two dosages (generally quite high) were employed, making it difficult to assess
the potential carcinogenic activity of lead compounds at relatively nontoxic concentrations.
It is also difficult to assess the true toxicity caused by these agents, since properly de-
signed toxicity studies were generally not performed in parallel with these cancer studies.
As shown in Table 12-19, lead nitrate produced no tumors in rats when given at very low
concentrations, but lead phosphate administered subcutaneously at relatively high levels in-
duced a high incidence of renal tumors in two studies. Lead powder administered orally re-
sulted in lymphomas and leukemia; when given intramuscularly only one fibrosarcoma was pro-
duced in 50 animals. Lead naphthenate applied as a 20 percent solution in benzene two times
each week for 12 months resulted in the development of four adenomas and one renal carcinoma
in a group of 50 mice (Baldwin et al., 1964). However, in this study control mice were not
12-231
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painted with benzene. Tetraethyl lead at 0.6 mg given in four divided doses between birth and
21 days to female mice resulted in 5 of 36 surviving animals developing lymphomas, while 1 of
26 males treated similarly and 1 of 39 controls developed lymphomas (Epstein and Mantel,
1968).
Lead subacetate has also been tested in the mouse lung adenoma bioassay (Stoner et al.,
1976). This assay measures the incidence of nodules forming in the lung of strain A/Strong
mice following parenteral administration of various test agents. Nodule formation in the lung
does not actually represent the induction of lung cancer but merely serves as a general meas-
ure of carcinogenic potency independent of lung tissue (Stoner et al., 1976). Lead subacetate
was administered to mice at 150, 75, and 30 mg (total dose), which represented the maximum
tolerated dose (MTD), 1/2 MTD, and 1/5 MTD, respectively, over a 30-week period using 15 sepa-
rate i.p. injections (Stoner et al., 1976). Survivals at the three doses were 15/20 (MTD),
12/20 (1/2 MTD), and 17/20 (1/5 MTD), respectively, with 11/15, 5/12, and 6/17 survivors
having lung nodules. Only at the highest doses was the incidence of nodules greater than in
the untreated mice. However, these authors concluded that on a molar-dose basis lead subace-
tate was the most potent of all the metallic compounds examined. Injection of 0.13 mmol/kg
lead subacetate was required to produce one lung tumor per mouse, indicating that this com-
pound was about three times more potent than urethane (at 0.5 mmol/kg) and approximately 10
times more potent than nickelous acetate (at 1.15 mmol/kg). The mouse lung adenoma bioassay
has been widely utilized for examining carcinogenic activity of chemical agents in experimen-
tal animals and is well recognized as a highly accurate test system for assessing potential
carcinogenic hazards of metals and their compounds (Stoner et al., 1976). Recent studies
utilizing the lung tumor bioassay in strain A mice have demonstrated that administration of
magnesium or calcium acetates along with lead subacetate eliminated the tumorigenic activity
of lead in this test system (Poirier et al., 1984). These results indicate that essential
divalent metals can protect against the carcinogenic effects of lead. Lead oxide combined
with benzopyrene administered intratracheally resulted in 11 adenomas and 1 adenocarcinoma in
a group of 15 hamsters, while no lung neoplasias were observed in groups receiving benzopyrene
or lead oxide alone (Kobayashi and Okamoto, 1974).
Administration of lead acetate to rats has been reported to produce other types of
tumors, e.g., testicular, adrenal, thyroidi pituitary, prostate, lung (Zawirska and Medras,
1968), and cerebral gliomas (Oyasu et al., 1970). However, in other animal species, such as
dogs (Azar et al., 1973; Fouts and Page, 1942) and hamsters (Van Esch and Kroes, 1969), lead
acetate induced either no tumors or only kidney tumors (Table 12-19).
The above studies seem to implicate some lead compounds as carcinogens in experimental
animals, but they were not designed to address the question of lead carcinogenesis in a defi-
nitive manner. In contrast, a study by Azar et al. (1973) examined the oncogenic potential of
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lead acetate at a number of doses and in addition monitored a number of toxicological para-
meters in the experimental animals. Azar et al. (1973) gave 0, 10, 50, 100, 500, 1000, and
2000 ppm dose levels of lead (as lead acetate) to rats during a two-year feeding study. Fifty
rats of each sex were utilized at doses of 10-500 ppm, while 100 animals of each sex were used
as controls. After the study was under way for a few months, a second 2-year feeding study
was initiated using 20 animals of each sex in groups given doses of 0, 1000, or 2000 ppm. The
study also included four male and four female beagle dogs at each dosage of lead ranging from
0 to 500 ppm in a 2-year feeding study. During this study, the clinical appearance and behav-
ior of the animals were observed, and food consumption, growth, and mortality were recorded.
Blood, urine, fecal, and tissue lead analyses were done periodically using atomic absorption
spectrophotometry. A complete blood analysis was done periodically, including blood cell
count, hemoglobin, hematocrit, stippled cell count, prothrombin time, alkaline phosphatase,
urea nitrogen, glutamic-pyruvate transaminase, and albumin-to-globulin ratio. The activity of
the enzyme delta-aminolevulinic acid dehydrase (ALA-D) in the blood and the excretion of its
substrate, delta-aminolevulinic acid (ALA), in the urine were also determined. A thorough ne-
cropsy, including both gross and histologic examination, was performed on all animals.
Table 12-20 depicts the mortality and incidence of kidney tumors reported by Azar et al.
(1973). At 500 ppm (0.05 percent) and above, male rats developed a significant number of re-
nal tumors. Female rats did not develop tumors except when fed 2000 ppm lead acetate. Two
out of four male dogs fed 500 ppm developed a slight degree of cytomegaly in the proximal con-
voluted tubule but did not develop any kidney tumors. The number of stippled erythrocytes in-
creased at 10 ppm in the rats but not until 500 ppm in the dogs. ALA-D was decreased at 50
ppm in the rats but not until 100 ppm in the dogs. Hemoglobin and hematocrit, however, were
not depressed in the rats until they received a dose of 1000 ppm lead. These results illus-
trate that the induction of kidney tumors coincides with moderate to severe toxicological
doses of lead acetate, for it was at 500-1000 ppm lead in the diet that a significant in-
crease in mortality occurred (see Table 12-20). At 1000 and 2000 ppm lead, 21-day-old wean-
ling rats showed no tumors but did show histological changes in the kidney comparable to those
seen in adults receiving 500 ppm or more lead in their diet. Also of interest from the Azar
et al. (1973) study is the direct correlation obtained in dogs between blood lead level and
kidney lead concentrations. A dietary lead level of 500 ppm produced a blood lead concentra-
tion of 80 pg/dl within 24 months, which corresponds to a level at which humans often show
clinical signs of lead poisoning (see Section 12.4.1). The kidney lead concentration corres-
ponding to this blood lead level was 2.5 |jg/g (wet weight), while at 50 pg/dl in blood the
kidney lead levels were 1.5 ug/g. Assuming similar pharmacokinetic distribution of lead in
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TABLE 12-20. MORTALITY AND KIDNEY TUMORS IN RATS FED LEAD ACETATE FOR TWO YEARS
Nominal (actual)
concentration in
ppm of Pb in diet
0 (5)
10 (18)
50 (62)
100 (141)
500 (548)
0 (3)
1000 (1130)
2000 (2102)
No. of rats
of each sex
100
50
50
50
50
20
20
20
%
Male
37
36
36
36
52
50
50
80
mortality
Female
34
30
28
28
36
35
50
35
% Kidney tumors
Male Female
0 o
0 0
0 0
0 0
10 0
0 0
50 0
80 35
Measured concentration of lead in diet.
Includes rats that either died or were sacrificed ui extremis.
Source: Azar et al. (1973).
the dogs as in rats, it can be stated that chronic exposure to 500 ppm of dietary lead, pro-
ducing prolonged elevation of blood lead to 80 ug/dl and resulting in a concentration of
2.5 MQ/g in the kidney can cause elevation in the incidence of kidney tumors.
Animal carcinogenesis studies conducted with lead and its compounds are numerous; how-
ever, with the exception of the study by Azar et al. (1973), they do not provide much useful
information. Most of the studies shown in Table 12-18 were conducted with only one lead com-
pound in one animal species, the rat. In cases where other lead compounds were tested or
where other animal species were used, only a single high dosage level was administered, and
parameters of toxicity such as those monitored in the Azar et al. (1973) study were not mea-
sured. Although it is clear from these studies as a whole that lead is a carcinogen in ex-
perimental animals, until more investigations such as that of Azar et al. (1973) are conducted
it is difficult to determine the relative carcinogenic potency of lead. There remains a need
to test thoroughly the carcinogenic activity of lead compounds in experimental animals. These
tests should include several modes of administration, many dosages spanning non-toxic as well
as toxic levels, and several different lead compounds or at least a comparison of a relatively
water-soluble form such as lead acetate with a less soluble form such as lead oxide.
12.7.2.3 Cell Transformation. Although cell transformation is an in vitro experimental sys-
tem, its end point is a neoplastic change. There are two types of cell transformation assays:
(1) those employing continuous cell lines; and (2) those employing cell cultures prepared from
embryonic tissue. Use of continuous cell lines has the advantage of ease in preparation of
12-234
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the cell cultures, but these cells generally have some properties of a cancer cell. The ab-
sence of a few characteristics of a cancer cell in these continuous cell lines allows for an
assay of cell transforming activity. End points include morphological transformation (ordered
cell growth to disordered cell growth), ability to form colonies in soft agar-containing
medium (a property characteristic of cancer cells), and ability of cells to form tumors when
inoculated into experimental animals. Assays that utilize freshly isolated embryonic cells
are generally preferred to those that use cell lines, because embryonic cells have not yet
acquired any of the characteristics of a transformed cell. The cell transformation assay
system has been utilized to examine the potential carcinogenic activity of a number of chemi-
cal agents, and the results seem to agree generally with the results of carcinogenesis tests
using experimental animals. Cell transformation assays can be made quantitative by assessing
the percentage of surviving colonies exhibiting morphological transformation. Verification of
a neoplastic change can be accomplished by cloning these cells and testing their ability to
form tumors in animals.
Lead acetate has been shown to induce morphological transformation in Syrian hamster em-
bryo cells following a continuous exposure to 1 or 2.5 pg/ml of lead in culture medium for
nine days (DiPaolo et al., 1978). The incidence of transformation increased from 0 percent in
untreated cells to 2.0 and 6.0 percent of the surviving cells, respectively, following treat-
ment with lead acetate. Morphologically transformed cells were capable of forming fibrosarco-
mas when cloned and administered to "nude" mice and Syrian hamsters, while no tumor growth re-
sulted from similar inoculation with untreated cells (DiPaolo et al., 1978). In the same
study, lead acetate was shown to enhance the incidence of simian adenovirus (SA-7) induction
of Syrian hamster embryo cell transformation. Lead acetate also caused significant enhance-
ment (~2- to 3- fold) at 100 and 200 ug/ml following three hours of exposure. In another
study (Casto et al., 1979), lead oxide also enhanced SA-7 transformation of Syrian hamster
embryo cells almost 4-fold at 50 pM following three hours of exposure (Casto et al., 1979).
The significance of enhanced virally induced carcinogenesis in relationship to the carcino-
genic potential of an agent is not well understood.
Morphological transformation induced by lead acetate was correlated with the ability of
the transformed cells to form tumors in appropriate hosts (see above), indicating that a truly
neoplastic change occurred in tissue culture. The induction of neoplastic transformation by
lead acetate suggests that this agent is potentially carcinogenic at the cellular level. How-
ever, with iji vitro systems such as the cell transformation assay it is essential to compare
the effects of other, similar types of carcinogenic agents in order to evaluate the response
and to determine the reliability of the assay. The incidence of transformation obtained with
lead acetate was greater than the incidence following similar exposure to NiCl2, but less than
12-235
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that produced by CaCr04 (Heck and Costa, 1982a). Both nickel and chromium have been impli-
cated in the etiology of human cancer (Costa, 1980). These results thus suggest that lead
acetate has effects similar to those caused by other metal carcinogens. In particular, the
ability of lead acetate to induce neoplastic transformation in cells in a concentration-depen-
dent manner is highly suggestive of potential carcinogenic activity. It should also be noted
that lead acetate induced these transformations at concentrations that decreased cell survival
by only 27 percent (DiPaolo et al., 1978). Further studies from other laboratories utilizing
the cell transformation assay and other lead compounds are needed.
12.7.3 Genotoxicity of Lead
Since cancer is known to be a disease of altered gene expression, numerous studies have
evaluated changes in DMA consequent to exposure to suspected carcinogenic agents. The general
response associated with such alterations in regulation of DMA function has been called geno-
toxicity. Various assay systems developed to examine specific changes in DNA structure and
function caused by carcinogenic agents include assays that evaluate chromosomal aberrations,
sister chromatid exchange, mutagenicity, and functional and structural features of DNA meta-
bolism. Lead's effects on these parameters are discussed below.
12.7.3.1 Chromosomal Aberrations. Two approaches have been used in the analysis of effects
of lead on chromosomal structure. The first approach involves culturing lymphocytes either
from humans exposed to lead or from experimental animals given a certain dosage of lead. The
second approach involves exposing cultured lymphocytes directly to lead. For present pur-
poses, emphasis will not be placed on the type of chromosomal aberration induced, since most
of the available studies do not appear to associate any specific type of chromosomal aberra-
tion with lead exposure. Little is known of the significance of chromosomal aberrations in
relationship to cancer, except that in a number of instances genetic diseases associated with
chromosomal aberrations often enhance the probability of neoplastic disease. However, impli-
cit in a morphologically distinct change in genetic structure is the possibility of an altera-
tion in gene expression that represents a salient feature of neoplastic disease.
Contradictory reports exist regarding the induction of chromosomal aberrations in lympho-
cytes from humans exposed to lead (Tables 12-21 and 12-22). These studies have been grouped
in two separate tables based upon their conclusions. Those studies reporting a positive ef-
fect of lead on chromosomal aberrations are indexed in Table 12-21, whereas studies reporting
no association between lead exposure and chromosomal aberrations are indexed in Table 12-22.
Unfortunately, these studies are difficult to thoroughly evaluate because of many unknown
variables (e.g., absence of sufficient evidence of lead intoxication, no dose-response rela-
tionship, and absence of information regarding lymphocyte culture time). To illustrate, in a
12-236
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TABLE 12-21. CYTOGENET1C INVESTIGATIONS OF CELLS FROM INDIVIDUALS EXPOSED TO LEAD: POSITIVE STUDIES
Number of Cell
exposed Number of culture
subjects controls time, hr
8 14 ?
10 10 72
14 5 48
105 - 72
11 - 68-70
(before and after
exposure)
44 15 72
23 20 48
20 20 46-48
26 (4 low, not 72
16 medium, given
6 high ex-
posure)
12 18 48-72
Lead level in
blood, ug/dl,
or urine, ug/£
62-89
(blood)
60-100
(blood)
155-720
(urine)
11.6-97.4
mean, 37.7
(blood)
34-64
(blood)
30-75
(blood)
44-95
(not given)
53-100
(blood)
22.5-65
(blood)
24-49
(blood)
Exposed subjects
Workers in a lead
oxide factory
Workers in a chem-
ical factory
Workers in a zinc
plant, exposed to
fumes & dust of
cadmium, zinc &
lead
Blast-furnace work-
ers, metal grin-
ders, scrap metal
processers
Workers in a
lead- acid battery
plant and a lead
foundry
Individuals in a
lead oxide fac-
tory
Lead-acid battery
Belters, tin workers
Ceramic, lead, &
battery workers
Sue Her workers
Electrical storage
battery workers
Type of damage
Chromatid and
chromosome
Chromatid gaps,
breaks
Gaps, fragments,
exchanges, dicen-
trics, rings
"Structural ab-
normalities,"
gaps, breaks,
hyperploidy
Gaps, breaks,
fragments
Chromatid and
chromosome
aberrations
Di Gentries,
rings, fragments
Breaks, frag-
ments
Gaps, chroma-
tid and chro-
mosome aberra-
tions
Chromatid and
chromosome aberra-
tions
Remarks
Increase in
chronosonal damage
correlated with
increased ALA
excretion
No correlation with
blood lead levels
Thought to be caused
by lead, not cadmium
or zinc
No correlation with
ALA excretion or
blood lead levels
No correlation
with ALA-D activity
in red cells
Positive correlation
with length of expo-
sure
Factors other than
lead exposure may be
required for severe
aberrations
Positive correlation
with blood lead levels
Positive correlation
with blood lead levels
I
References
Schwanitz et al.
(1970)
Gath & Thiess
(1972)
Oeknudt et al.
(1973)
Schwanitz et al.
(1975)
Forni et al.
(1976)
Garza-Chapa
et al. (1977)
Deknudt et al.
(1977b)
Sarto et al.
(1978)
Nordenson et al.
(1978)
Forni et al.
(1980)
Source: International Agency for Research on Cancer (1980), with codifications
-------
TABLE 12-22. CYTOGEHETIC INVESTIGATIONS OF CELLS FROM INDIVIDUALS EXPOSED TO LEAD: NEGATIVE STUDIES
NiMber of
exposed subjects
29
32
35
.*
j
j
3 24
9
30
NiMber of
control s
20
20
35
15
9
20
Cell culture
tine, hrs
46-48
46-48
45-48
48
72
4B
Blood lead
level , ug/dl
Not given, stated
to be 20-30%
higher than controls
Range not given;
highest level was
590 mg/1 [sic]
Control, <4; ex-
posed, 4 - >12
19.3 (lead)
0.4 (cadmium)
40 ± 5,
for 7 weeks
Control, 11.8-13.2;
exposed, 29-33
Exposed subjects
Policemen "permanently in
contact with high levels of
automotive exhaust"
Workers in lead manufacturing
industry; 3 had acute lead
intoxication
Shipyard workers employed as
"burners" cutting metal struc-
tures on ships
Mixed exposure to zinc, lead,
and cadmium in a zinc-smelting
plant; significant increase in
chromatid breaks and exchanges.
Authors suggest that cadmium
was the major cause of this
damage
Volunteers ingested capsules
containing lead acetate
Children living near a lead
smelter
References
Bauchinger et
(1972)
Schnid et al.
O'Riordan and
(1974)
Bauchinger et
(1976)
al.
(1972)
Evans
al.
Bulsw & De France
(1976)
Bauchinger et
(1977)
al.
Source: International Agency for Research on Cancer (1980).
-------
number of the studies where lead exposure correlated with an increased incidence of chromosom-
al aberrations (Table 12-21), lymphocytes were cultured for 72 hours. Most cytogenetic stu-
dies have been conducted with a maximum culture time of 48 hours to avoid high background
levels of chromosomal aberrations due to multiple cell divisions during culture. Therefore,
it is possible that the positive effects of lead on chromosomal aberrations may require the
longer culture period in order to be observed. Nonetheless, it is evident that in the negative
studies, the blood lead concentration was generally lower than in the studies reporting a
positive effect of lead on chromosomal aberrations, although in many of the latter instances
blood lead levels indicated severe exposure. In some of these positive studies there was a
correlation in the incidence of gaps, fragments, chromatid exchanges, and other chromosomal
aberrations with blood lead levels (Sarto et al., 1978; Nordenson et al., 1978). However, as
indicated in Table 12-21, in other studies there were no direct correlations between indices
of lead exposure (i.e., ALA excretion) and numbers of chromosomal aberrations. Nutritional
factors such as Ca2 levels HI vivo or iji vitro are also important since it is possible that
the effects of lead on cells may be antagonized by Ca2+ (Mahaffey, 1983; Poirier etal.,
1984). As is usually the case in studies of human populations exposed to lead, exposure to
other metals (zinc, cadmium, and copper) that may produce chromosomal aberrations was preva-
lent. These studies did not attempt to determine the specific lead compound to which the
individuals were exposed.
In a more recent study by Form' et al. (1980), 18 healthy females occupationally exposed
to lead were evaluated for chromosomal aberrations in their lymphocytes cultured for 48 or 72
hours. There were more aberrations at the 72-hour culture time compared with the 48-hour cul-
ture period in both control and lead-exposed groups, but this difference was not statistically
significant. However, statistically significant differences from the 72-hour controls were
noted in the 72-hour culture obtained from the lead exposed group. These results demonstrate
that the extended 72-hour culture time results in increased chromosomal aberrations in the
control lymphocytes and that the longer culture time was apparently necessary to detect the
effects of lead on chromosomal structure. However, the blood lead levels in the exposed fe-
males ranged from 24 to 59 (jg/dl, while control females had blood lead levels ranging from 22
to 37 ug/dl. The small effect of lead on chromosomal aberration may be due to the absence of
sufficient differences in the extent of lead exposure. Additionally, many agents that induce
chromosomal aberrations require extended time periods for the lesion to be expressed, as indi-
cated above for lead.
Some studies have also been conducted on the direct effect of soluble lead salts on cul-
tured human lymphocytes. In a study by Beek and Obe (1974), a longer (72-hr) culture time was
used and lead acetate was found to induce chromosomal aberrations at 100 uM. Lead acetate had
no effect on chromatid aberrations induced with X-rays or alkylating agents (Beek and Obe,
12-239
-------
1975). In another study (Deknudt and Deminatti, 1978), lead acetate at 1 and 0.1 mM caused
minimal chromosomal aberrations. Both cadmium chloride (CdCl2) and zinc chloride (2nCl2) were
more potent than lead acetate in causing these changes; however, both CdCl2 and ZnCl2 also
displayed greater toxicity than lead acetate.
Chromosomal aberrations have been demonstrated in lymphocytes from cynomolgus monkeys
treated chronically with lead acetate (6 mg/day, 6 days/week for 16 months), particularly when
they were kept on a low-calcium diet (Deknudt et al., 1977a). These aberrations accompanying a
low-Ca2 diet were characterized by the authors as severe (chromatid exchanges, dispiraliza-
tion, translocations, rings, and polycentric chromosomes). Similar results were observed In
mice (Deknudt and Gerber, 1979). The effect of low calcium on chromosomal aberrations induced
by lead could be due to interaction of Ca2 and Pb2 at the level of the chromosome (Mahaffey
1983). Leonard and his coworkers found no effect of lead on the incidence of chromosomal ab-
errations in accidentally intoxicated cattle (Leonard et al., 1974) or in mice given 1 g Pb/i
drinking water for 9 months (Leonard et al., 1973). However, Muro and Goyer (1969) found gaps
and chromatid aberrations in bone marrow cells cultured for four days after isolation from
mice that had been maintained on 1 percent dietary lead acetate for two weeks. Chromosomal
loss has been reported (Ahlberg et al., 1972) in Drosophila exposed to triethyl lead (4 mg/1)
but inorganic lead had no effect (Ramel, 1973). Lead acetate has also been shown to induce
chromosomal aberrations in cultured cells other than lymphocytes, viz. Chinese hamster ovary
cells (Bauchinger and Schmid, 1972).
These studies demonstrate that under certain conditions, lead compounds are capable of
inducing chromosomal aberrations jm vivo and in tissue cultures. The ability of lead to in-
duce these chromosomal changes appears to be concentration-dependent and highly influenced by
calcium levels. In lymphocytes isolated from patients or experimental animals, relatively
long (72-hr) culture conditions are required for the abnormalities to be expressed, indicating
a requirement for cellular processes (e.g., DNA repair) to interact with the hidden lead-
induced DNA lesions to produce a morphologically manifested aberration.
12.7.3.2 Sister Chromatid Exchange. Sister chromatid exchange affords a means of visually
assessing the normal movement of DNA in the genome. The sister chromatid exchange assay offers
a very sensitive probe for the effects of genotoxic compounds on DNA rearrangement, as a
number of chemicals with carcinogenic activity are capable of increasing these exchanges
(Sandberg, 1982). The effect of lead on such movement has been examined in cultured lympho-
cytes (Beek and Obe, 1975), with no increase in exchanges observed at lead acetate concentra-
tions of 0.01 mM. Two more recent studies have examined the effect of human lead exposure on
the incidence of sister chromatid exchange in peripheral blood lymphocytes (Dalpra et al.
1983; Grandjean et al., 1983). A study by Dalpra et al. (1983) involved an investigation of
the incidence of sister chromatid exchanges in 19 children who lived in a widely contaminated
12-240
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area and who showed Increased lead absorption. Blood lead levels as well as erythrocyte ALA-D
activity and zinc protoporphyrin in blood were measured in the exposed group as well as in the
12 controls. The exposed group had blood lead levels ranging from 29.3 to 62.7 ug/dl and
ALA-D activity ranging from 8 to 32 erythrocytes. In contrast, the control group had blood
lead values ranging from 10 to 21 ug/dl and ALA-D values ranging from 36 to 78/ml erythro-
cytes. Similarly, zinc protoporphyrin ranged from 65 to 341 ug/dl in the mil/ml exposed group
and from 9 to 38 ug/dl in the controls. Thus, the population studied was well defined with
regard to their exposure to lead. Based upon the measured parameters, the lead exposure was
not severe. The results of an examination of sister chromatid exchange frequencies indicated
no significant differences between the control group and the lead-exposed group. This appears
to be a well-conducted study with excellent documentation of lead exposure in the population.
The study examined the same exposed children that were observed by Form' et al. (1981) in
their study of chromosomal aberrations. Forni et al. (1981), however, found an increased
level of chromosomal aberration after 48- or 72-hr culture times (vide supra), suggesting that
chromosomal aberrations may appear in the absence of detectable sister chromatid exchange
(Dalpra et al., 1983). These findings emphasize the importance of utilizing a battery of test
systems with different endpoints to accurately comprehend the true genotoxic potential of an
agent.
Another recent well-conducted study, by Grandjean et al. (1983), examined the incidence
of sister chromatid exchange in adult lead-exposed men. There was a significant correlation
(p <0.001) between the observed zinc protoporphyrin levels and the incidence of sister chro-
matid exchange in the lead-exposed group. However, there was a poor correlation between blood
lead levels and sister chromatid exchange, which suggested that zinc protoporphyrin levels
were a better indicator of lead exposure than blood lead levels in this study. Interestingly,
during a 4-week cessation of lead exposure, the elevated incidence of sister chromatid ex-
change diminished, together with the zinc protoporphyrin levels and blood lead levels in per-
fused blood lymphocytes. The persistence of the sister chromatid exchange depends to a large
extent upon the proliferation and half-life of the lymphocyte. In workers newly exposed to
lead for four months there were clear increases in lead exposure parameters in the absence of
any increase in the sister chromatid exchange frequency. Collectively, this study demon-
strates for the first time a positive correlation between lead exposure and sister chromatid
exchange. Further, it indicates that the increased sister chromatid exchange is not rapid in
its induction, since it was only observed in lymphocytes after chronic exposure. Additional-
ly, in lymphocytes the increased sister chromatid exchange was reversed when the lead exposure
was decreased. It should be noted, however, that these effects occurred in only one cell type
and the incidence of sister chromatid exchange may be uniquely different for every cell type
12-241
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jn vivo. Nevertheless, these results support the potential injurious role lead may have on
chromosomal structure and function.
The ability of agents such as lead to cause abnormal rearrangements in the structure of
DNA, as revealed by the appearance of chromosomal aberrations and sister chromatid exchanges
has become an important focus in carcinogenesis research. Current theories suggest that can-
cer may result from an abnormal expression of oncogenes (genes that code for protein products
associated with virally induced cancers). Numerous oncogenes are found in normal human DMA
but the genes are regulated such that they are not expressed in a carcinogenic fashion. Re-
arrangement of these DMA sequences within the genome is associated with ontogenesis, although
the activation of oncogenes has not been demonstrated to be the cause of tumor induction
Evidence has been presented suggesting that chromosomal aberrations such as translocations are
associated with certain forms of cancer and with the movement of oncogenes in regions of the
DNA favoring their expression in cancer cells (Shen-Ong et al., 1982). By inducing aberra-
tions in chromosomal structure, lead may enhance the probability of an oncogenic event.
12.7.3.3 Effects on Bacterial and Mammalian Mutagenesis Systems. Bacterial and mammalian
mutagenesis test systems examine the ability of chemical agents to induce changes in DNA
sequences of a specific gene product that is monitored by selection procedures. They measure
the potential of a chemical agent to produce a change in DNA, but this change is not likely to
be the same alteration in gene expression that occurs during oncogenesis. However, if an
agent affects the expression of a particular gene product that is being monitored, then It
could possibly affect other sequences that may result in cancer. Since many carcinogens are
also mutagens, it is useful to employ such systems to evaluate genotoxic effects of lead.
Use of bacterial systems for assaying metal genotoxicity must await further development
of bacterial strains that are appropriately responsive to known mutagenic metals (Rosenkranz
and Poirier, 1979; Simmon, 1979; Simmon et al., 1979; Nishioka, 1975; Nestmann et al., 1979).
Mammalian cell mutagenic systems that screen for specific alterations in a defined gene muta-
tion have not been useful in detecting mutagenic activity with known carcinogenic metals (Heck
and Costa, 1982b). In plants, however, chromosomal aberrations in root tips (Mukherji and
Maitra, 1976) and other mutagenic activity, such as chlorophyll mutations (Reddy and Vaidya-
nath, 1978) and reproductive organ mutations (Lower et al., 1983), have been demonstrated with
lead.
12.7.3.4 Effects on Parameters of DNA Structure and Function. There are a number of very
sensitive techniques for examining the effect of metals on DNA structure and function in in-
tact cells. Although these techniques have not been extensively utilized with respect to
metal compounds, future research will probably be devoted to this area. Considerable work has
been done to understand the effects of metals on enzymes involved in DNA replication.
12-242
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SI rover and Loeb (1976) examined effects of lead and other metal compounds upon the
fidelity of replication of DNA by a viral DNA polymerase. High concentrations of metal ions
(1n some cases in the millimolar range) were required to decrease the fidelity of replication,
but there was a good correlation between metal ions that are carcinogenic or mutagenic and
their activity in decreasing the fidelity of DNA replication. This assay system measures the
ability of a metal ion to incorporate incorrect (non-homologous) bases using a defined poly-
nucleotide template. In an intact cell, this would cause the induction of a mutation if the
Insertion of an incorrect base is phenotypically expressed. Since the interaction of metal
ions with cellular macromolecules is relatively unstable, misincorporation of a base during
semi-conservative DNA replication or during DNA repair synthesis following the induction of a
DNA lesion with a metal could alter the base sequence of the DNA in an intact cell. Lead at
4 mM was among the metals listed as mutagenic or carcinogenic that caused a decrease in the
fidelity of replication (Sirover and Loeb, 1976). Other metals active in decreasing fidelity
included: Ag+, Be2*, Cd2+, Co2"1", Cr2+, O3+, Cu2+, Mn2+, and Ni2+. Metals that decreased
fidelity are metals also implicated as carcinogenic or mutagenic (Sirover and Loeb, 1976).
In a similar study, Hoffman and Niyogi (1977) demonstrated that lead chloride was the
most potent of 10 metals tested in inhibiting RNA synthesis (i.e., Pb2+ > Cd2+ > Co2+ > Mn2+ >
L1+ > Na > K ) for both types of templates tested, i.e., calf thymus DNA and T4 phage DNA.
These results were explained in terms of the binding of these metal ions more to the bases
than to the phosphate groups of the DNA (i.e., Pb2+ > Cd2+ > Zn2* > Mn2* > Mg2+ > Li+ = Na+ =
K+). Additionally, metal compounds, such as lead chloride, with carcinogenic or mutagenic
activity were found to stimulate mRNA chain initiation at 0.1 mM concentrations.
These well-conducted mechanistic studies provide evidence that lead can affect a molecu-
lar process associated with normal regulation of gene expression. Although far removed from
the intact cell situation, these effects suggest that lead may be genotoxic. In a related
study, lead sulfate along with numerous other toxic and carcinogenic metals was shown to cause
an S-phase specific cell cycle block (Costa et al., 1982). A significant effect of lead was
observed at 20 \M. These results indicate that this metal will interfere with the normal syn-
thesis and replication of DNA. A recent study has examined the ability of lead acetate to in-
duce strand breaks and DNA repair synthesis in cultured mammalian cells (Robinson et al.,
1984). Lead acetate was slightly more potent than NiCl2 in inducing true DNA single strand
breaks, based upon neutral nucleoid gradient analysis, but was considerably less potent than
CaCr04 or HgCl2 (Robinson et al., 1984). Lead acetate also caused induction of DNA repair
synthesis based upon analysis with CsCl equilibrium density gradient sedimentation. DNA
repair synthesis was elevated about 10-fold above the control level at 200 uM lead acetate
exposure for 1 hr (Robinson et al., 1984). These results further support the concept that
lead can have effects upon the DNA.
12-243
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12.7.4 Lead as an Initiator and Promoter of Carcinogenesis
An agent may act as a carcinogen in two distinct ways: (1) as an initiator; or (2) as a
promoter (Weisburger and Williams, 1980). By definition, an initiator must be able to inter-
act with DNA to produce a genetic alteration, whereas a promoter acts in a way that allows the
expression of an altered genetic change responsible for cancer. Since lead is capable of
transforming cells directly in culture and affecting DNA-to-DNA and ONA-to-RNA transcription,
it may have some initiating activity. Its ability to induce chromosomal aberrations is also
indicative of initiating activity. The ability of lead to induce proliferation in the kidney
as indicated by increased DNA, RNA, and protein synthesis suggests that it may also have pro-
moting activity in cancer target tissues (Choie and Richter, 1974a,b). Its similarity to Ca2+
suggests that it may alter regulation of this cation in processes (e.g., cell growth) related
to promotion (see Section 12.3.5). A recent study, demonstrating that subsequent administra-
tion of basic lead acetate greatly enhances the development of renal tubular cell tumors in
rats previously treated with n-ethyl-n-hydroxyethylnitrosamine, indicates a promotional role
of this agent as well (Hiasa et al., 1983). Thus, evidence is accumulating to suggest that
lead and its compounds are complete carcinogens possessing both initiating and promoting
activity.
12.7.5 Summary and Conclusions
It is evident from studies reviewed above that, at relatively high concentrations, lead
displays some carcinogenic activity in experimental animals such as the rat. Lead may act
either as an initiator or promoter of carcinogenic activity, because it has genotoxic pro-
perties related to cancer initiation, as well as cellular effects related to the promotion or
expression of cancer. The presence of intranuclear lead inclusion bodies in the kidney may
pertain to lead's carcinogenic effects, since both the formation of these bodies and the in-
duction of tumors occur at relatively high doses of lead. Evidence exists for the presence of
these inclusion bodies in kidneys from experimental animals treated with lead and also in one
well documented human case report of renal cancer associated with excessive lead exposure.
The interaction of lead with key non-histone chromosomal proteins in the nucleus to form the
inclusion bodies or the presence of inclusion bodies in the nucleus may alter genetic func-
tion, thus leading to cell transformation. Obviously, elucidating the mechanism of lead car-
cinogenesis requires further research efforts and only theories can be formulated regarding
its oncogenic action at present.
It is hard to draw clear conclusions concerning what role lead may play in the induction
of human neoplasia. Epidemiological studies of lead-exposed workers provide no definitive
findings. However, statistically significant elevations in respiratory tract and digestive
system cancer in workers exposed to lead and other agents warrant concern. Also, since lead
12-244
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acetate can produce renal tumors in some experimental animals, it may be prudent to assume
that lead compounds may be carcinogenic in humans, as was concluded by the International
Agency for Research on Cancer (1980). However, this statement is qualified by noting that
lead has been observed to increase tumorigenesis rates in animals only at relatively high
concentrations, and therefore it does not appear to be a potent carcinogen. A recent epidemi-
ological study and two case reports suggest the possible association of lead exposure with
the induction of kidney tumors in humans; however, several other epidemiological studies have
not thus far demonstrated a significant excess of kidney tumors in lead workers. In vitro
studies further support the genotoxic and carcinogenic role of lead, but also indicate that
lead is not potent in these systems either.
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12.8 EFFECTS OF LEAD ON THE IMMUNE SYSTEM
12.8.1 Development and Organization of the Immune System
Component cells of the immune system arise from a pool of pluripotent stem cells in the
yolk sack and liver of the developing fetus and in the bone marrow and spleen of the adult.
Stem cell differentiation and maturation follows one of several lines to produce lymphocytes
macrophages, and polymorphonuclear leukocytes. These cells have important roles in immunolog-
ical function and host defense.
The predominant lymphocyte class develops in the thymus, which is derived from the third
and fourth pharyngeal pouches at 9 weeks of gestation in man (day 9 in mice). In the thymus
microenvironment, they acquire characteristics of thymus-derived lymphocytes (T-cells), then
migrate to peripheral thymic-dependent areas of the spleen and lymph nodes. T-cells are
easily distinguished from other lymphocytes by genetically defined cell surface markers that
allow them to be further subdivided into immunoregulatory helper and suppressor T-cells.
T-cells also participate directly as cytolytic effector cells against virally infected host
cells, malignant cells, and foreign tissues, as well as in delayed-type hypersensitivity (DTH)
reactions where they elaborate lymphokines that modulate the inflammatory response. T-cells
are long-lived lymphocytes and are not readily replaced. Thus, any loss or injury to T-cells
may be detrimental to the host and may result in increased susceptibility to viral, fungal
bacterial, or parasitic diseases. Individuals with acquired immune deficiency syndrome (AIDS)
are examples of individuals with T-cell dysfunction. There is ample evidence that depletion
by environmental agents of thymocytes or stem cell progenitors during lymphoid organogenesls
can produce permanent immunosuppression.
The second major lymphocyte class differentiates from a lymphoid stem-cell in a yet un-
defined site in man, which would correspond functionally to the Bursa of Fabricius in avian
species. In man, B-lymphocyte maturation and differentiation probably occur embryologically
in gut-associated lymphoid tissue (GALT) and fetal liver, as well as adult spleen and bone
marrow. This is followed by the peripheral population of thymic-independent areas of spleen
and lymph nodes. Bone marrow-derived lymphocytes (B-cells), which mature independently of the
thymus, possess, specific immunoglobulin receptors on their surfaces. The presence of cell
surface immunoglobulin (slg) at high density is the major characteristic separating B-cells
from T-cells. Following interaction with antigens and subsequent activation, B-lymphocytes
proliferate and differentiate into antibody-producing plasma cells. In contrast to the
long-lived T-cell, B-cells are rapidly replaced by newly differentiating stem cells.
Therefore, lesions in the B-cell compartment may be less serious than those in the T-cell
compartment since they are more easily reversed. Insult to B-cells at the stem cell or
terminal maturation stage can result in suppression of specific immunoglobulin and enhanced
susceptibility to infectious agents whose pathogenesis is limited by antibodies.
12-246
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Pluripotent stem cells also give rise to lymphocytes whose lineages are still unclear.
Some possess natural cytolytic activity for tumor cells (natural killer cell activity), while
others, devoid of T- and B-cell surface markers (null cells), participate in antibody-depen-
dent cell-mediated cytotoxicity (ADCC). The pluripotent stem cell pool also contains precur-
sors of monocyte-macrophages and polymorphonuclear leukocytes (PMN). The macrophage has a
major role in presentation and processing of certain antigens, in cytolysis of tumor target
cells, and in phagocytosis and lysis of persistent intracellular infectious agents. Also, it
actively phagocytizes and kills invading organisms. Defects in differentiation or function of
PMNs or macrophages predispose the host to infections by bacteria and other agents.
This introduction should make it evident that the effects of an element such as lead on
the immune system may be expressed in complex or subtle ways (see also Section 12.3.5.3.5).
In some cases, lead might produce a lesion of the immune system not resulting in markedly
adverse health effects, especially if the lesion did not occur at an early stem cell stage or
during a critical point in lymphoid organogenesis. On the other hand, some lead-induced
immune system effects might adversely affect health through increasing susceptibility to
infectious agents or neoplastically transformed cells if, for example, they were to impair
cytocidal or bactericidal function.
12.8.2 Host Resistance
One way of ascertaining if a chemical affects the immune response of an animal is to
challenge an exposed animal with a pathogen such as an infectious agent or oncogen. This pro-
vides a general approach to determine if the chemical interferes with host immune defense
mechanisms. Host defense is a composite of innate immunity, part of which is phagocyte activ-
ities, and acquired immunity, which includes B- and T-lymphocyte and enhanced phagocyte reac-
tivities. Analysis of host resistance constitutes a holistic approach. However, dependent on
the choice of the pathogen, host resistance can be evaluated somewhat more selectively.
Assessment of host resistance to extracellular microbes such as Staphylococci, Salmonella
typhimurium, Escherichia coli, or Streptococcus pneumoniae and to intracellular organisms such
as Listen'a monocytogenes or Candida albicans primarily measures intact humoral immunity and
cell-mediated immunity, respectively. Immune defense to extracellular organisms requires
T-lymphocyte, B-lymphocyte, and macrophage interactions for the production of specific anti-
bodies to activate the complement cascade and to aid phagocytosis. Antibodies can also
directly neutralize some bacteria and viruses. Resistance to intracellular organisms requires
T-lymphocyte and macrophage interactions for T-lymphocyte production of lymphokines, which
further enhance immune mechanisms including macrophage bactericidal activities. An additional
T-lymphocyte subset, the cytolytic T-cell, is involved in resistance to tumors; immune
defenses against tumors are also aided by NK- and K-lymphocytes and macrophages.
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12.8.2.1 Infectivity Models. Numerous studies designed to assess the influence of lead on
host resistance to infectious agents consistently have shown that lead impairs host resis-
tance, regardless of whether the defense mechanisms are predominantly dependent on humoral- or
cell-mediated immunity (Table 12-23).
TABLE 12-23. EFFECT OF LEAD ON HOST RESISTANCE TO INFECTIOUS AGENTS
Species
Mouse
Rat
Rat
Mouse
Mouse
Mouse
Mouse
Infectious agent
S. typhimurium
E. coli
S. epidermidis
L. monocytogenes
EMCt virus
EMC virus
Langat virus
Lead dose
200 ppm
2 mg/100 g
body weight
2 mg/100 g
body weight
80 ppm
2000 ppm
13 ppm
50 mg/kg
body weight
Lead exposure
i.p.
i.v.
i.v.
oral
oral
oral
oral
9
5
iy
iy
iy
iy
30 days
1 day
1 day
; 4 wk
; 2 wk
; 10 wk
; 2 wk
Mortality*
54%
96%
80%
100%
100%
80%
68%
(13%)
(0%)
(0%)
(0%)
(19%)
(50%)
(0%)
Reference
Hemphil
Cook et
Cook et
1 et al. (1971)
al. (1975)
al
. (1975)
Lawrence (1981a)
Gainer (1977b)
Exon et
al
Thind and
. (1979)
Khan (1978)
The percent mortality is reported for the lowest dose of lead in the study that significantly
altered host resistance. The percent mortality in parentheses is that of the non-lead-treated,
infected control group.
tEMC = encephalomyocarditis virus.
Mice (Swiss Webster) injected i.p. for 30 days with 100 or 250 ug (per 0.5 ml) of lead
nitrate and inoculated with Salmonella typhimurium had higher mortality (54 and 100 percent,
respectively) than non-lead-injected mice (13 percent) (Hemphill et al., 1971). These concen-
trations of lead, by themselves, did not produce any apparent toxicity. Similar results were
observed in rats acutely exposed to lead (one i.v. dose of 2 mg/100 g body weight) and chal-
lenged with Escherichia coli (Cook et al., 1975). In these two studies, lead could have in-
terfered with the clearance of endotoxin from the S^ typhimurium or E. coli. and the animals
may have died from endotoxin shock, and not septicemia, due to the lack of bacteriostatic or
bactericidal activities. However, the study by Cook et al. (1975) also included a non-
endotoxin-producing gram-positive bacterium, Staphylococcus epidermidis, and lead still
impaired host resistance. In another study, lead effects on host resistance to the intra-
cellular parasite Listen'a monocytogenes were monitored (Lawrence, 1981a). CBA/J mice orally
exposed to 16, 80, 400, and 2000 ppm lead for four weeks were assayed for viable Listerja
after 48 and 72 hours, and for mortality after 10 days. Only 2000 ppm lead caused significant
12-248
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inhibition of early bactericidal activity (48-72 hr), but 80-2000 ppm lead produced 100 per-
cent mortality, compared with 0 percent mortality in the 0-16 ppm lead groups. Other reports
have suggested that host resistance is impaired by lead exposure of rodents. Salaki et al.
(1975) indicated that lead lowered resistance of mice to Staphylococcus aureus, Listen'a, and
Candida, and observed higher incidence of inflammation of the salivary glands in lead-exposed
rats (Grant et al., 1980) may be due, in part, to lead-induced increased susceptibility to
infections.
Inhalation of lead has also been reported to lower host resistance to bacteria.
Schlipkb'ter and Frieler (1979) exposed NMRI mice to an aerosol of 13-14 ug/m3 lead chloride
and clearance of Serratia marcesens in the lungs was reduced significantly. Microparticles of
lead in lungs of mice were also shown to lower resistance to Pasteurella multocida, in that
6 ug of lead increased the percentage of mortality by 27 percent (Bouley et al., 1977).
Lead has also been shown to increase host susceptibility to viral infections. CD-I mice,
administered 2,000 and 10,000 ppm lead in drinking water for two weeks and subsequently inocu-
lated with encephalomyocarditis (EMC) virus, had a significant increase in mortality (100 per-
cent at 2,000 ppm; 65 percent at 10,000 ppm) compared with control EMC virus-infected mice (13
percent) (Gainer, 1977b). In another study (Exon et al., 1979), Swiss Webster mice were ex-
posed to 13, 130, 1300, or 2600 ppm lead for 10 weeks in their drinking water and were infec-
ted with EMC virus. Although as low as 13 ppm lead caused a significant increase in mortality
(80 percent) in comparison with the non-lead-treated EMC virus-infected mice (50 percent),
there were no dose-response effects, in that 2600 ppm lead resulted in only 64 percent mortal-
ity. The lack of a dose-response relationship in the two studies with EMC virus (Gainer,
1977b; Exon et al., 1979) suggests that the higher doses of lead may directly inhibit EMC
Infectivity as well as host defense mechanisms. Additional studies have confirmed that lead
inhibits host resistance to viruses. Mice treated orally with lead nitrate (10-50 mg/kg/ day)
for two weeks had suppressed antibody titers to Langat virus (Type B arbovirus) and increased
titers of the virus itself (Thind and Singh, 1977), and the lead-inoculated, infected mice had
higher mortalities (25 percent at 10 mg/kg; 68 percent at 50 mg/kg) than the non-1ead-infected
mice (0 percent) (Thind and Khan, 1978).
The effects of lead on bacterial and viral infections in humans have never been studied
adequately; there is only suggestive evidence that human host resistance may be lowered by
lead. Children with persistently high blood lead levels who were infected with Shigella
enteritis had prolonged diarrhea (Sachs, 1978). In addition, lead workers with blood lead
levels of 22-89 ug/dl have been reported to have more colds and influenza infections per year
(Ewers et al., 1982). This study also indicated that secretory IgA levels were suppressed
significantly in lead workers with a median blood lead level of 55 ^g/dl. Secretory IgA is a
major factor in immune defense against respiratory as well as gastrointestinal infections.
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Hicks (1972) points out that there is need for systematic epidemiological studies on the
effects of elevated lead levels on the incidence of infectious diseases in humans. The cur-
rent paucity of information precludes formulation of any clear dose-response relationship for
humans. Epidemiological investigations may help to determine if lead alters the immune system
of man and consequently increases susceptibility to infectious agents and neoplasia.
12.8.2.2 Tumor Models and Neoplasia. The carcinogenicity of lead has been studied both as a
direct toxic effect of lead (see Section 12.7) and as a means of better understanding the
effects of lead on the body's defense mechanisms. Studies by Gainer (1973, 1974) demonstrated
that exposure of CD-I mice to lead acetate potentiated the oncogenicity of a challenge with
Rauscher leukemia virus (RLV), resulting in enhanced splenomegaly and higher virus titers in
the spleen presumably through an immunosuppressive mechanism. Recent studies by Kerkvliet and
Baecher-Steppan (1982) revealed that chronic exposure of C57BL/6 mice to lead acetate in
drinking water at 130-1300 ppm enhanced the growth of primary tumors induced by Moloney sar-
coma virus (MSV). Regression of MSV-induced tumors was not prevented by lead exposure, and
lead-treated animals resisted late sarcoma development following primary tumor resistance.
Depressed resistance to transplantable MSV tumors was associated with a reduced number of
macrophages, which also exhibited reduced phagocytic activity.
In addition to enhancing the transplantability of tumors or the oncogenicity of leukemia
viruses, lead has also been shown to facilitate the development of chemically induced tumors.
Kobayashi and Okamoto (1974) found that intratracheal dosing of benzo(a)pyrene (BaP) combined
with lead oxide resulted in an increased frequency of lung adenomas and adenocarcinomas over
hamsters exposed to BaP alone. Similarly, exposure to lead acetate enhanced the formation of
N(4'-fluoro-4-biphenyl) acetamide-induced renal carcinomas from 70 to 100 percent and reduced
the latency to tumor appearance (Hinton et al., 1980). Recently, Koller et al. (1985) found
that exposure to lead (2600 ppm in drinking water) for 18 months increased the frequency of
tumors, predominantly renal carcinomas, in rats. Similarly, Schrauzer et al. (1981) found
that adding lead at 5 ppm to drinking water of C3H/St mice infected with Bittner milk factor
diminished the uptake of selenium and reduced its anticarcinogenic effects, causing mammary
tumors to appear at the same high incidence as in selenium-unsupplemented controls. Lead
likewise significantly accelerated tumor growth and shortened survival in this model.
The above studies on host susceptibility to various pathogens, including infectious
agents and tumors, indicate that lead could be detrimental to health by methods other than di-
rect toxicity. In order to understand the mechanisms by which lead suppresses host resistance
maintained by phagocytes, humoral immunity, and/or cell-mediated immunity, the immune system
must be dissected into its functional components and the effects of lead on each, separately
and combined, must be examined in order that the mechanism(s) of the immunomodulatory poten-
tial of lead can be understood.
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12.8.3 Humoral Immunity
12.8.3.1 Antibody liters. A low antibody 'titer in animals exposed to lead could explain the
increased susceptibility of animals to extracellular bacteria and some viruses (see Table
12-24), as well as to endotoxins (Selye et al., 1966; Filkins, 1970; Cook et al., 1974;
Schumer and Erve, 1973; Rippe and Berry, 1973; Truscott, 1970). Specific antibodies can
directly neutralize pathogens, activate complement components to induce lysis, or directly or
indirectly enhance phagocytosis via Fc or C3 receptors, respectively. Studies in animals and
humans have assayed the effects of lead on serum immunoglobulin levels, specific antibody
levels, and complement levels. Analysis of serum immunoglobulin levels is not a good measure
of specific immune reactivity, but it would provide evidence for an effect on B-lymphocyte
development.
TABLE 12-24. EFFECT OF LEAD ON ANTIBODY TITERS
Species
Antigen
Lead dose and
exposure
Effect on
antibody titer
Reference
Rabbit Pseudorabies virus
Rat S. typhimurium
2,500 ppm; 10 wk
Decrease Koller (1973)
5,000-20,000 ppm; 3 wk Decrease Stankovic and Jugo
(1976)
Rat
Bovine serum albumin 10-1,000 ppm; 10 wk
Mouse Sheep erythrocytes
0.5-10 ppnT; 3 wk
Decrease Koller et al.
(1983)
Decrease Blakley et al.
(1980)
aLead was administered as tetraethyl lead; other studies used inorganic forms.
Lead had little effect on the serum immunoglobulin levels in rabbits (Fonzi et al.,
1967a), children with blood lead levels of 40 ug/dl (Reigart and Graber, 1976), or lead
workers with 22-89 ug/dl (Ewers et al., 1982). On the other hand, most studies have shown
that lead significantly impairs antibody production. Acute oral lead exposure (50,000 ppm/kg)
produced a decreased titer of anti-typhus antibodies in rabbits immunized with typhus vaccine
(Fonzi et al., 1967b). In New Zealand white rabbits challenged with pseudorabies virus, lead
(oral exposure to 2500 ppm for 70 days) caused a 9-fold decrease in antibody titer to the
virus (Koller, 1973). However, lead has not always been shown to reduce titers to virus.
Vengris and Mare (1974) did not observe depressed antibody titers to Newcastle disease virus
in lead-treated chickens, but their lead treatment was only for 35 days prior to infection.
Lead-poisoned children also had normal anti-toxoid titers after booster immunizations with
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tetanus toxoid (Reigart and Graber, 1976). In another study, Wistar rat dams were exposed to
5,000, 10,000, or 20,000 ppm lead for 20 days following parturition (Stankovic and Jugo,
1976). The progeny were weaned at 21 days of age and given standard laboratory chow for an
additional month. At that time, they were injected with Salmonella typhimurium. and serum
antibody titers were assessed. Each dosage of lead resulted in significantly reduced antibody
titers. More recently, rats (Sprague-Dawley) given 10 ppm lead acetate orally for 10 weeks
had a significant suppression in antibody titers when challenged with bovine serum albumin
(BSA) and compared with USA-immunized non-lead-exposed rats (Keller et al., 1983). Develop-
ment of a highly sensitive, quantitative, enzyme-linked immunosorbent assay (ELISA) contrib-
uted to detecting the immunosuppressive activity of lead at this dosage.
Tetraethyl lead also has been responsible for reduced antibody titers in Swiss-cross mice
(Blakley et al., 1980). The mice were exposed orally to 0.5, 1.0, and 2.0 ppm tetraethyl lead
for 3 weeks. A significant reduction in hemagglutination titers to sheep red blood cells
(SRBC) occurred at all levels of exposure.
12.8.3.2 Enumeration of Antibody Producing Cells (Plaque-Forming Cells). From the above re-
sults, it appears that lead inhibits antibody production. To evaluate this possible effect at
the cellular level, the influence of lead on the number of antibody-producing cells after pri-
mary or secondary immunization can be assessed. In primary humoral immune responses (mostly
direct), IgM plaque-forming cells (PFC) are measured, whereas in secondary or anamnestic
responses (mostly indirect), IgG PFC are counted. The primary immune response represents an
individual's first contact with a particular antigen. The secondary immune response repre-
sents re-exposure to the same antigen weeks, months, or even years after the primary antibody
response has subsided. The secondary immune response is attributed to persistence, after
initial contact with the antigen, of a substantial number of antigen-sensitive memory cells.
Impairment of the memory response, therefore, results in serious impairment of humoral immun-
ity in the host.
Table 12-25 summarizes the effects of lead on IgM or IgG PFC development. Mice exposed
orally to tetraethyl lead (0.5, 1, or 2 ppm) for three weeks exhibited a significant reduction
in the development of IgM and IgG PFC (Blakley et al., 1980). Mice (Swiss Webster) exposed
orally to 13, 137, or 1375 ppm inorganic lead for eight weeks had reduced numbers of IgM PFC
in each lead-exposed group (Keller and Kovacic, 1974). Even the lowest lead group (13 ppm)
had a decrease. The secondary response (IgG PFC, induced by a second exposure to antigen SRBC
seven days after the primary immunization) was inhibited to a greater extent than the primary
response. This study indicated that chronic exposure to lead produced a significant decrease
in the development of IgM PFC and IgG PFC. When Swiss Webster mice were exposed to 13, 130,
and 1300 ppm lead for 10 weeks and hyperimmunized by SRBC injections at week 1, 2, and 9, the
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TABLE 12-25. EFFECT OF LEAD ON THE DEVELOPMENT OF ANTIBODY-PRODUCING CELLS
Species
Mouse
Mouse
Mouse
Mouse
Mouse
Rat
Antigen*
SRBC (in vivo)
SRBC On vivo)
SRBC (in vivo)
SRBC (in vivo)
SRBC (in vivo)
SRBC (Tn vTtro + 2-ME)
SRBC (in vivo)
Lead dose and exposure
13-1370 ppm; 8 wk
0.5-2 ppm tetraethyl lead;
3 wk
13-1370 ppm; 10 wk
4 mg (i.p. or orally)
16-2000 ppm; 1-10 wk
16-80 ppm; 4 wk
2000 ppm; 4 wk
25-50 ppm; pre/postnatal
Effectt
IgM PFC (D)
IgG PFC (D)
IgM PFC (D)
IgG PFC (D)
IgG PFC (D)
IgM PFC (I)
IgG PFC (D)
IgM PFC (N)
IgM PFC (I)
IgM PFC (D)
IgM PFC (D)
Reference
Keller and
Kovacic
(1974)
Blakley et al .
(1980)
Koller and
Roan (1980a)
Koller et al .
(1976)
Lawrence
(1981a)
Luster et al .
(1978)
Mouse
Mouse
SRBC (in vitro)
SRBC (Tri vvtro + 2-ME)
50-1000 ppm; 3 wk
50-1000 ppm; 3 wk
SRBC (iji vitro + 2-ME) 2-20 ppm (in vitro)
IgM PFC (D)
IgM PFC
(N or I)
IgM PFC (I)
Blakley and
Archer (1981)
Lawrence
(1981b,c)
*The antigenic challenge with sheep red blood cells (SRBC) was in vivo or in vitro after in
yiyo exposure to lead unless otherwise stated. The jjn vitro assays were performed in the
presence or absence of 2-mercaptoethanol (2-ME).
tlgM/G PFC = immunoglobulin M/G plaque-forming cells; D = decreased response; N = unaltered
response; I = increased response.
memory response as assessed by the enumeration of IgG PFC was significantly inhibited at 1300
ppm (Keller and Roan, 1980a). This suggests that the temporal relationships between lead
exposure and antigenic challenge may be critical. Other studies support this interpretation.
Female Sprague-Dawley rats with pre- and post-natal exposure to lead (25 or 50 ppm) had a
significant reduction in IgM PFC (Luster et al., 1978). In contrast, CBA/J mice exposed
orally to 16-2000 ppm lead for 1-10 weeks did not have altered IgM PFC responses to SRBC
(Lawrence, 1981a). Furthermore, when Swiss Webster mice were exposed to an acute lead dose (4
mg lead orally or i.p.), the number of IgG PFC was suppressed, but the number of IgM PFC was
enhanced (Koller et al., 1976).
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The influence of lead on the development of RFC in mice was assessed further by iji vivo
exposure to lead, removal of spleen cells, and jn vitro analysis of RFC development. Initi-
ally it appeared that low doses of lead (16 and 80 ppm) enhanced development, and only a high
dose (2000 ppm) inhibited the development of IgM RFC (Lawrence, 1981a). However, a later
study by Blakley and Archer (1981) indicated that 50-1000 ppm lead consistently inhibited IgM
RFC. Through the analysis of mixed cultures of lead-exposed lymphocytes (nonadherent cells)
and unexposed macrophages (adherent cells), and vice versa, as well as of ±n vitro responses
to antigens that do not require macrophage help (i.e., lipopolysaccharide, IPS), their data
indicated that the effects of lead may be at the level of the macrophage. This was substan-
tiated by the fact that 2-mercaptoethanol (2-ME), a compound that can substitute for at least
one macrophage activity, was able to reverse the inhibition by lead. This may explain why rn
vivo lead exposure (16 and 80 ppm) appeared to enhance the in vitro IgM RFC responses in the
study by Lawrence (1981a), because 2-ME was present in the j_n vitro assay system. Further-
more, ui vitro exposure to lead (2 or 20 ppm) in spleen cell cultures with 2-ME enhanced the
development of IgM RFC (Lawrence, 1981b,c).
These experiments indicate that lead modulates the development of antibody-producing
cells as well as serum antibody titers, which supports the notion that lead can suppress hu-
moral immunity. However, it should be noted that the dose and route of exposure of both lead
and antigen may influence the modulatory effects of lead. The adverse effects of lead on hu-
moral immunity may be due more to lead's interference with macrophage antigen processing
and/or antigen presentation to lymphocytes than to direct effects on B-lymphocytes. These
mechanisms require further investigation.
12.8.4 Cell-Mediated Immunity
12.8.4.1 Delayed-Type Hypersensitivity. T-lymphocytes (T-helper and T-suppressor cells) are
regulators of humoral and cell-mediated immunity as well as effectors of two aspects of cell-
mediated immunity. T-cells responsive to delayed-type hypersensitivity (DTH) produce lym-
phokines that induce mononuclear infiltrates and activate macrophages, which are aspects of
chronic inflammatory responses. In addition, another subset of T-cells, cytolytic T-cells,
cause direct lysis of target cells (tumors or antigenically modified autologous cells) when in
contact with the target. To date, the effects of lead on cytolytic T-cell reactivity have not
been measured, but the influence of lead on inducer T-cells has been studied (Table 12-26).
Groups of mice injected i.p. daily for 30 days with 13.7-137 ppm lead were subsequently
sensitized i.v. with SRBC. The DTH reaction was suppressed in these animals in a dose-related
fashion (MUller et al., 1977). The secondary DTH response was inhibited in a similar fashion.
In another study (Faith et al., 1979), the effects of chronic low-level pre- and postnatal
lead exposure on cellular immune functions in Sprague-Dawley rats were assessed. Female rats
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TABLE 12-26. EFFECT OF LEAD ON CELL-MEDIATED IMMUNITY
Species
Mouse
Rat
Mouse
Mouse
Lead dose and exposure
13.7-137 ppm; 4 wk
25-50 ppm; 8 wk
13-1300 ppm; 10 wk
16-2000 ppm; 4 wk
Parameter*
DTH
DTH
MLC
MLC
Effect
Decrease
Decrease
None
Decrease
Reference
Muller et al. (1977)
Faith et al. (1979)
Keller and Roan (19805)
Lawrence (1981a)
*DTH - delayed-type hypersensitivity; MLC = mixed lymphocyte culture.
were exposed to 25 or 50 ppm lead acetate continuously for seven weeks before breeding and
through gestation and lactation. The progeny were weaned at three weeks of age and continued
on the respective lead exposure regimen of their mothers for an additional 14-24 days. Thymic
weights and DTH responses were significantly decreased by both lead dosages. These results
Indicate that chronic low levels of lead suppress cell-mediated immune function.
The jn vitro correlate of the analysis of DTH responsive T-cells jn vivo is the analysis
of mixed lymphocyte culture (MLC) responsive T-cells. When two populations of allogeneic lym-
phoid cells are cultured together, cellular interactions provoke blast cell transformation and
proliferation of a portion of the cultured cells (Cerottini and Brunner, 1974; Bach et al.,
1976). The response can be made one-way by irradiating one of the two allogeneic prepara-
tions, in which case the irradiated cells are the stimulators (allogeneic B-cells and macro-
phages) and the responders (T-cells) are assayed for their proliferation. The mixed lympho-
cyte reaction is an im vitro assay of cell-mediated immunity analogous to \n vivo host versus
graft reactions.
Mice (DBA/2J) fed 13, 130, or 1300 ppm lead for 10 weeks were evaluated for responsive-
ness in mixed lymphocyte cultures. The 130-ppm lead dose tended to stimulate the lymphocyte
reaction, although no change was observed at the other dose levels (Koller and Roan, 1980b).
In another study (Lawrence, 1981a), mice (CBA/J) were fed 16, 80, 400, or 2000 ppm lead for
four weeks. The 16 and 80 ppm doses slightly stimulated, while the 2000 ppm dose suppressed,
the mixed lymphocyte reaction. It is important to note that in these i_n vitro MLC assays,
2-ME was present in the culture medium, and the 2-ME may have reversed the jm vivo effects of
lead, as was observed for the jn vitro PFC responses (Blakley and Archer, 1981).
The data on the effects of lead on humoral and cell-mediated immunity indicate that vn
vivo lead usually is immunosuppressive, but additional studies are necessary to fully under-
stand the temporal and dose relationship of lead's immunomodulatory effects. The jn vitro
12-255
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analysis of immune cells exposed to lead HI vivo suggest that the major cell type modified is
the macrophage; the suppress!ve effects of lead may be readily reversed by thiol reagents
possibly acting as chelators.
12.8.4.3 Interferon. Interferons (IF) are a family of low-molecular-weight proteins which
exhibit antiviral activity in sensitive cells through processes requiring new cellular RNA and
protein synthesis (Stewart, 1979). it has been speculated that tfie enhanced susceptibility Of
lead'treated mice to infectious virus challenge might be due to a decreased capacity of these
animals to produce viral or immune interferons or to respond to them. Studies by Gainer
(1974, 1977a) appeared to resolve this question and indicated that exposure of CD-I mice to
lead does not inhibit the antiviral action of viral IF iji vivo or jn vitro. In the later of
the two studies, lead exposure inhibited the protective effects of the IF inducers Newcastle
disease virus and poly I:poly C against encephalomyocarditis virus (EMC)-induced mortality.
These data suggest that, although lead did not directly interfere with the antiviral activity
of interferon, it might suppress viral IF production in vivo. Recently, Blakley et al. (1982)
re-examined this issue and found that female BDFj mice exposed to lead-acetate in drinking
water at concentrations ranging from 50 to 1000 jjg/ml for three weeks produced amounts of IF
similar to controls given a viral IF inducer, Tilorone. Similarly, the jji vitro induction of
immune IF by the T-cell mitogens—phytohemagglutinin, concanavalin A, and staphylococcal
enterotoxin--in lymphocytes from lead-exposed mice were unaltered compared with controls
(Blakley et al., 1982). Thus, lead exposure does not appear to significantly alter the lym-
phocyte's ability to produce immune interferon. Therefore, it must be assumed that increased
viral susceptibility associated with chronic lead exposure in rodents is by mechanisms other
than interference with production of or response to interferon.
12.8.5 Lymphocyte Activation by Mitogens
Mitogens are lectins that induce activation, blast-cell transformation, and proliferation
in resting lymphocytes. Certain lectins bind specifically to (1) T-cells (i.e., phytohemag-
glutinin [PHA] and concanavalin A [Con A]), (2) B-cells (i.e., lipopolysaccharide [IPS] of
gram-negative bacteria), or (3) both (i.e., pokeweed mitogen [PWM]). The resulting blasto-
genic response can be used to assess changes in cell division of T- and B-lymphocytes. The
biological significance of the following studies is difficult to interpret since exposure to
lead was either jjn vivo or ir\ vitro at different doses and for different exposure periods.
12.8.5.1 In Vivo Exposure. Splenic lymphocytes from Swiss Webster mice exposed orally to
2000 ppm lead for 30 days had significantly depressed proliferative responses to PHA (Table
12-27) which were not observed after 15 days of exposure (Gaworski and Sharma, 1978). Sup-
pression was likewise observed with PWM, a T- and B-cell mitogen. These observations with
12-256
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TABLE 12-27. EFFECT OF LEAD EXPOSURE ON MITOGEN ACTIVATION OF LYMPHOCYTES
ro
cn
Species
Mice
Mice
Rats
Mice
Mice
Mice
Mice
Lead dose and exposure
In vivo, 250 and 2000
ppm, 30 days
In vivo, 13, 130, and 1300
pp«, 10 weeks
In vivo, pre/postnatal
25 and 50 ppn, 7 weeks
In vivo. 0.08-10 *H, 4 weeks
In vivo, 1300 ppa, 8 weeks
In vivo, 50, 200, and 1000 ppa,
T~weeks
In vitro. 10"*-10"6 M for
Mitogen3
PHA (T-Cell)
PWH (T and B-Cell)
Con A fT-Cell)
LPS (B-Cell)
Con A
PHA
Con A, PHA
LPS
Con A, PHA
LPS
Con A, PHA, SEA
LPS
Con A, PHA
Effect
Significantly depressedhat
2000 ppm on day 30 only
Significantly depressed at
2000 pee on both days 15
and 30°
No effect
No effect
Significantly depressed at
25 and 50 ppn
Significantly depressed at
BO ppn only
No effect
Depressed at 2 and 10 wH
Significantly depressed
No effect
Increased to allc
No effect
Slightly increased at
Reference
Gaworski and
Shanw (1978)
Roller et al. (1979)
Faith et aJ. (1979)
Lawrence (1981c)
Neil an et al. (1980)
Biakley and Archer (1982)
Lawrence (1981a,b)
Mice In vitro. 0.1, 0.5, 1.0
Tor full culture period
Mice In vitro. 10"3-10"7 M
Tor 72 hours
LPS
PHA
PWM
LPS
highest dose at day 2, no
effect at day 3.5 .
Increased up to 245JT
Increased at all doses by
up to 453X°
Increased by approximately
250X at 0.1 and 0.5 nM only
Increased by up to 312%
Gaworski and
Shama (1978)
Shenker et al. (1977);
Gallagher et al.
-------
T-cell mitogens were confirmed in Sprague-Daw'ley rats exposed orally to 25 or 50 ppm lead pre-
and postnatally for seven weeks (Faith et al., 1979). Splenic T-cell responses to Con A and
PHA were significantly diminished. A similar depression of Con A and PHA responses occurred
in lymphocytes from C57B1/6 mice exposed to 1300 ppm lead for 8 weeks (Neilan et al., 1980).
Lead impaired blastogenic transformation of lymphocytes by both T-cell mitogens, although the
B-cell proliferative response to IPS was not impaired.
In contrast to reports that lead exposure suppressed the blastogenic response of T-cells
to mitogens, several laboratories have reported that lead exposure does not suppress T-cell
proliferative responses (Koller et al., 1979; Lawrence, 1981c; Blakley and Archer, 1982).
These differences are not easily reconciled since analysis of the lead dose employed and ex-
posure period (Table 12-26) provides little insight into the observed differences in T-cell
responses. In one case, a dose of 2000 ppm for 4 weeks produced a clear depression, while a
lesser dose of 1300 ppm produced no effect at 10 weeks in another laboratory. These data are
confusing and may reflect technical differences in performing the T-cell blastogenesis assay
in different laboratories, a lack of careful attention to lectin response kinetics, or the
influence of suppressor macrophages. Thus, no firm conclusion can be drawn regarding the
ability of jj] vivo exposure to lead to impair the proliferative capacity of T-cells.
The blastogenic response of B-cells to LPS was unaffected in four different _ui vivo stud-
ies at lead exposure levels of 25-1300 ppm (Roller et al., 1979; Faith et al., 1979; Neilan
etal., 1980; Blakley and Archer, 1982). Lawrence (1981c), however, reported that the LPS
response was suppressed after 4 weeks exposure at 2 and 10 mM lead. The weight of the data
suggests that the proliferative response of B-cells to LPS is probably not severely impaired
by lead exposure.
12.8.5.2 In Vitro Exposure. The biological relevance of immunological studies in which lead
was added in vitro to normal rodent splenocytes in the presence of a mitogen (Table 12-27) is
questionable since differences probably reflect either a direct toxic or stimulatory effect by
the metal. These models may, however, provide useful information regarding metabolic and
functional responses in lymphocytes using lead as a probe.
In one study, lymphocytes were cultured in the presence of lead (10 , 10 , and 10 M).
A slight but significant increase in lymphocyte transformation occurred on day 2 at the high-
est lead dosage when stimulated with Con A or PHA (Lawrence, 1981b). In a follow-up study
where the kinetics of the lectin response were examined (Lawrence, 1981a), lead (10~ , 10" ,
and 10" M) significantly suppressed the Con A- and PHA-induced proliferative responses of
lymphocytes on day 2, but not on days 3-5. In yet another jm vitro exposure study, lympho-
cytes cultured in the presence of 0.1, 0.5, or 1.0 mM lead had a significantly enhanced
response to PHA (Gaworski and Sharma, 1978). It should be kept in mind when considering these
12-258
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jn vitro exposure observations that lead has been demonstrated to be directly mitogenic to
lymphocytes (Shenker et al., 1977). The data discussed here suggest that lead may also be
slightly co-mitogenic with T-cell mitogens. Direct exposure of lymphocytes in culture to lead
can also result in decreased lymphocyte viability (Gallagher et al., 1979). In vitro studies
on the effect of lead on the B-cell blastogenic response to IPS indicated that lead is
potently co-mitogenic with LPS and enhanced the proliferative response of B-cells by 245 per-
cent (Lawrence 1981b,c) to 312 percent (Shenker et al., 1977; Gallagher et al., 1979).
12.8.6 Macrophage Function
The monocyte/macrophage is involved with phagocytosis, bactericidal activity, processing
of complex antigens for initiation of antibody production, interferon production, endotoxin
detoxification, and immunoregulation. Since some of these functions are altered in lead-
treated rodents (Table 12-28), the monocyte/macrophage or comparable phagocytic cell in the
liver has been suggested as a possible cellular target for lead (Trejo et al.} 1972; Cook et
al., 1974; MUller et al., 1977; Luster et al., 1978; Blakley and Archer, 1981).
Several laboratories have shown that a single i.v. injection of lead impaired the phago-
cytic ability of the reticuloendothelial system (RES) (Trejo et al., 1972; Cook et al., 1974;
Filkins and Buchanan, 1973). Trejo et al. (1972) found that an i.v. injection of 5 mg lead
impaired vascular clearance of colloidal carbon that resulted from an impaired phagocytic
ability of liver Kupffer cells. Similarly, others have confirmed that lead injected i.v. de-
pressed intravascular clearance of colloidal carbon (Filkins and Buchanan, 1973) as well as a
radiolabeled lipid emulsion (Cook et al., 1974). Opposite effects on RES function have been
seen when lead was given orally (Keller and Roan, 1977). Similarly, Schlick and Friedberg
(1981) noted that a 10-day exposure to 10-1000 pg lead enhanced RES clearance and endotoxin
hypersensitivity.
Lead has likewise been demonstrated to suppress macrophage-dependent immune responses
(Blakley and Archer, 1981). Exposure of BDFt mice to lead (50 ppm) for three weeks in drink-
ing water suppressed jm vitro antibody RFC responses to the macrophage-dependent antigens,
sheep red blood cells or dinitrophenyl-Ficoll, but not to the macrophage-independent antigen,
E. coli lipopolysaccharide. The macrophage substitute, 2-mercaptoethanol, and macrophages
from non-exposed mice restored lead-suppressed response. Castranova et al. (1980) found that
cultured rat alveolar macrophages exposed to lead had depressed oxidative metabolism.
The effects of heavy metals on endotoxin hypersensitivity were first observed by Selye et
al. (1966), who described a 100,000-fold increase in bacterial endotoxin sensitivity in rats
given lead acetate. The increased sensitivity to endotoxin was postulated to be due to a
blockade of the RES. Filkins (1970) subsequently demonstrated that endotoxin detoxification
12-259
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TABLE 12-28. EFFECT OF LEAD ON MACROPHAGE AND RETICULOENDOTHELIAL SYSTEM FUNCTION
Species
Lead dose
and mode
Parameter
Effect
Reference
Rat
Rat
Mouse
ro
i
Guinea Pig
Rat
Mouse
Mouse
2.25
single intravenous
injection
5 mg,
single intravenous
injection
13, 130, 1300 ppm
oral, 10-12 weeks
_3 _6
10 -10 M, in vitro
10" -10" M, in vitro
50-1000 ppm oral,
3 weeks
10-1000 pg,
10 days, intravenous
injection
Vascular clearance;
lipid emulsion
endotoxin sensitivity
Vascular clearance;
colloidal carbon
endotoxin sensitivity
Phagocytosis
Macrophage migration
Macrophage oxygen
metabolism
Plague-forming cell
response to macrophage
dependent antigens
Vascular clearance
Depressed
Increased
Depressed
Increased
Depressed
Depressed
Depressed
Depressed
Enhanced at
10 days;
no effect
at >30 days
Cook et al. (1974);
Trejo et al. (1972)
Trejo et al. (1972);
Filkins and
Buchanan (1973)
Kerkvliet and Baecher-
Steppan (1982)
Kiremidjian-
Schumacher et al. (1981)
Castranova et al. (1980)
Blakley and Archer
(1981)
Schlick and
Friedberg (1981)
Endotoxin sensitivity
Increased
-------
1s primarily a hepatic macrophage-mediated event that is profoundly impaired by lead exposure
(Trejo and Di Luzio, 1971; Filkins and Buchanan, 1973). The several types of data described
above suggest that macrophage dysfunction may be contributing to impairment of immune func-
tion, endotoxin detoxification, and host resistance following lead exposure.
12.8.7 Mechanisms of Lead Immunomodulation
The mechanism of toxic action of lead on cells is complex (see Section 12.2). Since lead
has a high affinity for sulfhydryl groups, a likely subcellular alteration accounting for the
immunomodulatory effects of lead on immune cells is its association with cellular thiols.
Numerous studies have indicated that surface and intracellular thiols are involved in lympho-
cyte activation, growth, and differentiation. Furthermore, the study by Blakley and Archer
(1981) suggests that lead may inhibit the macrophage's presentation of stimulatory products to
the lymphocytes. This process may rely on cellular thiols since the inhibitory effects of
lead can be overcome by an exogenous thiol reagent. Goyer and Rhyne (1973) have indicated
that lead ions tend to accumulate on cell surfaces, thereby possibly affecting surface recep-
tors and cell-to-cell communication. A study by Koller and Brauner (1977) indicated that lead
does alter C3b binding to its cell surface receptor.
12.8.8 Summary
Lead renders animals highly susceptible to endotoxins and infectious agents. Host sus-
ceptibility and the humoral immune system appear to be particularly sensitive. As postulated
in recent studies, the macrophage may be the primary immune target cell of lead. Lead-induced
immunosuppression occurs at low dosages that induce no evident toxicity and, therefore, may be
detrimental to the health of animals and perhaps of humans. The data accumulated to date pro-
vide good evidence that lead affects immunity, but additional studies are necessary to eluci-
date the actual mechanism by which lead exerts its immunosuppressive action. Knowledge of
the effects of lead on the immune system of man is lacking and must be properly ascertained
in order to determine permissible levels for human exposure. However, since this chemical
affects immunity in laboratory animals and is immunosuppressive at very low dosages, its
potential serious effects in man should be carefully considered.
12-261
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12.9 EFFECTS OF LEAD ON OTHER ORGAN SYSTEMS
12.9.1 The Cardiovascular System
Lead has long been reported to be associated with cardiovascular effects, at least at
high exposure levels. Some of the older literature bearing on this subject is reviewed here
In addition, because one of the the best understood pathophysiologic mechanisms of hyperten-
sion in humans is that resulting from renal disease, some clinical evidence linking lead-
induced renal effects to hypertension is discussed in Section 12.5.3.5. (A more detailed
discussion of lead-hypertension relationships, focusing on recently completed studies, can be
found in an Addendum to this document.)
In regard to some of the older literature on lead's cardiovascular effects, Dingwall-
Fordyce and Lane (1963) reported a marked increase in the cerebrovascular mortality rate amona
heavily exposed lead workers as compared with the normal expected rate. These workers were
exposed to lead during the first quarter of this century when working conditions were quite
bad. There has been no similar increase in the mortality rate reported for men employed In
recent times.
Cardiovascular structural and functional changes have been noted for both adults and
children with acute lead poisoning, but to date the extent of such studies has been limited
For example, cases have been described with regard to effects on the myocardium of children
always with clinical signs of poisoning. There is, of course, the possibility that the co-
existence of lead poisoning and myocarditis is coincidental; but in many cases in which en-
cephalopathy was present, the electrocardiographic abnormalities disappeared with chelatlon
therapy, suggesting that lead may have indeed been the original etiological factor (Freeman
1965; Myerson and Eisenhauer, 1963; Silver and Rodriguez-Torres, 1968). Silver and Rodriguez-
Torres (1968) noted abnormal electrocardiograms in 21 of 30 children (70 percent) having symp-
toms of lead toxicity. After chelation therapy, the electrocardiograms remained abnormal In
only four (13 percent) of the patients. In a review of five fatal cases of lead poisoning In
young children, degenerative changes in heart muscle were reported to be the proximate cause
of death (Kline, 1960). It is not clear that such morphological changes are a specific re-
sponse to lead intoxication. Kdsmider and Petelenz (1962) examined 38 adults over 46 years of
age with chronic lead poisoning. They found that 66 percent had electrocardiographic changes
a rate that was four times the expected rate for that age group.
Electron microscopy of the myocardium of lead-intoxicated rats (Asokan, 1974) and mice
(Khan et al., 1977) has shown diffuse degenerative changes. The susceptibility of the myo-
cardium to toxic effects of lead was supported by i_n vitro studies in rat mitochondria by Parr
and Harris (1976). These investigators found that the rate of Ca2 removal by rat heart
mitochondria is decreased by 1 nmol Pb/mg protein. Kopp and coworkers have demonstrated de-
pression of contractility, isoproterenol responsiveness, and cardiac protein phosphorylatlon
12-262
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(Kopp et al., 1980a), as well as high-energy phosphate levels (Kopp et al., 1980b) in hearts
of lead-fed rats. Similarly, persistent increased susceptibility to norepinephrine-induced
arrhythmias has been observed in rats fed lead during the first three weeks of life
(Hejtmancik and Williams, 1977, 1978, 1979a,b; Williams et al., 1977a,b).
The cardiovascular effects of lead in conjunction with cadmium have been studied in rats
following chronic low-level exposure by Perry and coworkers (Perry and Erlanger, 1978; Perry
et al., 1979; Kopp et al., 1980a,b). Perry and Erlanger (1978) exposed female weanling Long-
Evans rats to cadmium, lead, or cadmium plus lead (as acetate salts) at concentrations of 0.1,
1.0, or 5.0 ppm in deionized drinking water for up to 18 months. These authors reported sta-
tistically significant increases in systolic blood pressure for both cadmium and lead in the
range of 15-20 mm Hg. Concomitant exposure to both cadmium and lead usually doubled the
pressor effects of either metal alone. A subsequent study (Kopp et al., 1980a) using weanling
female Long-Evans rats exposed to 5.0 ppm cadmium, lead, or lead plus cadmium in deionized
drinking water for 15 or 20 months showed similar pressor effects of these two metals, alone
or in combination, on systolic blood pressure. Electrocardiograms performed on these rats
demonstrated statistically significant prolongation of the mean PR interval. Bundle elec-
trograms also showed statistically significant prolongations. Other parameters of cardiac
function were not markedly affected. Phosphorus-31 nuclear magnetic resonance (NMR) studies
conducted on perchloric acid extracts of liquid nitrogen-frozen cardiac tissue from these ani-
mals disclosed statistically significant reductions in adenosine triphosphate (ATP) levels and
concomitant increases in adenosine diphosphate (ADP) levels. Cardiac glycerol 3-phosphoryl-
choline (GPC) was also found to be significantly reduced using this technique, indicating a
general reduction of tissue high-energy phosphates by lead or cadmium. Pulse-labeling studies
using 32P demonstrated decreased incorporation of this isotope into myosin light-chain (LC-2)
in all lead or cadmium treatment groups relative to controls.
The results of this group of studies indicate that prolonged low-dose exposure to lead
(and/or cadmium) reduces tissue concentrations of high-energy phosphates in rat hearts and
suggest that this effect may be responsible for decreased myosin LC-2 phosphorylation and
subsequent reduced cardiac contractility. Other experiments by these authors (Kopp et al.,
1980b) were also conducted on isolated perfused hearts of weanling female Long-Evans rats
exposed to cadmium, lead, or lead plus cadmium in deionized drinking water at concentrations
of 50 ppm for 3-15 months. Incorporation of 32P into cardiac proteins was studied following
perfusion on inotropic perfusate containing isoproterenol at a concentration of 7 x 10 M.
Data from these studies showed a statistically significant reduction in cardiac active tension
in hearts from cadmium- or lead-treated rats. Incorporation of 32P was also found to be sig-
nficantly reduced in myosin LC-2 proteins. The authors suggested that the observed decrease
12-263
-------
in LC-2 phosphorylation could be involved in the observed decrease in cardiac active tension
in lead- or cadmium-treated rats.
There are conflicting reports regarding whether lead can cause atherosclerosis in experi-
mental animals. Scroczynski et al. (1967) observed increased serum lipoprotein and choles-
terol levels and cholesterol deposits in the aortas of rats and rabbits receiving large doses
of lead. On the other hand, Prerovska" (1973), using similar doses of lead given over an even
longer period of time, did not produce atherosclerotic lesions in rabbits.
Makasev and Krivdina (1972) observed a two-phase change in the permeability of blood ves-
sels (first increased, then decreased permeability) in rats, rabbits, and dogs that received a
solution of lead acetate. A phase change in the content of catecholamines in the myocardium
and in the blood vessels was observed in subacute lead poisoning in dogs (Mambeeva and
Kobkova, 1969). This effect appears to be a link in the complex mechanism of the cardiovas-
cular pathology of lead poisoning.
12.9.2 The Hepatic System
The effect of lead poisoning on liver function has not been extensively studied. In a
study of 301 workers in a lead-smelting and refining facility, Cooper et al. (1973) found an
increase in serum glutamic oxaloacetic transaminase (SGOT) activity in 11.5 percent of
subjects with blood lead levels below 70 MS/dl, in 20 percent of those with blood lead levels
of about 70 |jg/dl, and in 50 percent of the workers with blood lead levels of about 100 pg/dl.
The correlation (r = 0.18) between blood lead levels and SGOT was statistically significant.
However, there must also have been exposure to other metals, e.g., cadmium, since there was a
zinc plant in the smelter. In lead workers with moderate effects on the hematopoietic system
and no obvious renal signs, SGOT was not increased compared with controls on repeated examina-
tions (Hammond et al., 1980). In most studies on lead workers, tests for liver function are
not included.
The liver is the major organ for the detoxification of drugs. In Section 12.3.1.3 it is
mentioned that exposure to lead may cause altered drug detoxification rates as a result of in-
terference with the formation of heme-containing cytochrome P-450, which is part of the
hepatic mixed function oxidase system. This enzyme system is involved in the hepatic bio-
transformation of medicaments, hormones, and many environmental chemicals (Remmer et al.,
1966). Whereas a decrease in drug-metabolizing activity clearly has been demonstrated in
experimental animals given large doses of lead resulting in acute toxicity, the evidence for
effects of that type in humans is less consistent. Alvares et al. (1975) studied the effect
of lead exposure on drug metabolism in children. There was no difference between two normal
12-264
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children and eight children with biochemical signs of lead toxicity as far as their capacity
to metabolize two test drugs, antipyrine and phenylbutazone. In two acutely poisoned chil-
dren in whom blood levels of lead exceeded 60 ug/dl, antipyrine half-lives were significantly
longer than normal, and therapy with EDTA led to biochemical remission of the disease and
restoration of deranged drug metabolism toward normal. One of the "normal" children in this
study had a blood lead level of 40 ug/dl, but normal ALA-D and EP values. No data were given
on the analytical methods used for indices of lead exposure. Furthermore, the age of the
children varied from 1 to 7.5 years, which is significant because, as pointed out by the
authors, drug detoxification is age-dependent.
Meredith et al. (1977) demonstrated enhanced hepatic metabolism of antipyrine in lead-
exposed workers (PbB: 77-195 ug/dl) following chelation therapy. The significance of this
evidence of restored hepatic mixed oxidase function is, however, unclear because the pretreat-
ment antipyrine biologic half-life and clearance were not significantly different in lead-
exposed and control subjects. Moreover, there were more heavy smokers among the lead-exposed
workers than controls. Smoking increases the drug-metabolizing capacity and may thus counter-
act the effects of lead. Also, the effect of chelation on antipyrine metabolism in non-
exposed control subjects was not determined.
Hepatic drug metabolism was studied by Alvares et al. (1976) in eight adult patients
showing marked effects of chronic lead intoxication on the erythropoietic system. The plasma
elimination rate of antipyrine, which, as noted above, is a drug primarily metabolized by he-
patic microsomal enzymes, was determined in eight subjects prior to and following chelation
therapy. In seven of eight subjects, chelation therapy shortened the antipyrine half-lives,
but the effect was minimal. The authors concluded that chronic lead exposure results in sig-
nificant inhibition of the heme biosynthetic pathway without causing significant changes in
enzymatic activities associated with hepatic cytochrome P-450.
A confounding factor in the above three studies may be that treatment with EDTA causes an
increase in the glomerular filtration rate (GFR) if it has been reduced by lead (Section
12.5.3.3). This may cause a decrease in the half-lives of drugs. There are, however, no data
on the effect of chelating agents on GFR in children or adults with moderate signs of lead
toxicity.
In 11 children with blood lead levels between 43 and 52 ug/dl, Saenger et al. (1981)
found a decrease in 24-hour urinary 6-beta-hydroxycortisol excretion that correlated closely
(r = 0.85, p <0.001) with a standardized EDTA lead-mobilization test (1000 mg EDTA/m2 body
surface area). This glucocorticoid metabolite is produced by the same hepatic microsomal
mixed-function oxidase system that hydroxylates antipyrine. The authors suggest that the de-
pression of 6-beta-hydroxylation of cortisol in the liver may provide a non-invasive method
for assessing body lead stores in children (Saenger et al., 1981).
12-265
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In a few animal studies, special attention has been paid to morphological effects of lead
on the liver. Ledda-Columbano et al. (1983) investigated hepatic cell proliferation in male
Wistar rats given 1,4, or 9, i.v. injections of lead nitrate (5 umol/100 g body weight) at
10-day intervals. Although body weight was not significantly reduced and liver cell deaths
did not increase, liver weight and DNA activity were both significantly increased. This work
confirmed earlier findings by Columbano et al. (1983) that a single dose of lead nitrate (10
(jmo1/100 g) stimulated hepatic DNA synthesis in rats. Although the authors noted that cell
proliferation could have been due to an adaptive mechanism, such proliferation could also have
significant implications for liver carcinogenesis. More recently these findings were repli-
cated and extended by Dessi et al. (1984), who observed a twofold increase in relative liver
weight in rats 48 hours after an i.v. injection of lead nitrate (10 umol/100 g). The investi-
gators also found various indications of significantly increased cholesterol synthesis and
glucose-6-phosphate dehydragenase, both of which were seen as consistent with the hyperplasia
induced by lead.
White (1977) gave eight beagle dogs oral doses of lead carbonate, 50-100 mg Pb/kg b.w.
for 3-7 weeks. Lead concentrations were not measured in blood or tissues. Morphological
changes were noted in two dogs exposed from 5 weeks of age to 50 mg/kg. Changes in enzyme
activities were found in most exposed animals; for example, some dehydrogenases showed in-
creased activity after short exposure and decreased activity after longer exposures, mainly in
animals with weight losses. The small number of animals and the absence of data on lead con-
centrations makes it impossible to use these results for risk evaluations.
Hoffmann et al. (1974) noted moderate to marked morphological changes in baboon livers
after a single intravenous injection of large doses of lead acetate (25 mg/kg b.w.). It can
be concluded that effects on the liver may be expected to occur only at high exposure levels.
If effects on more sensitive systems, viz., the nervous and hematopoietic systems, are pre-
vented, no adverse effects should be noted in the liver.
12.9.3 The Gastrointestinal System
Colic is usually a consistent early symptom of lead poisoning, warning of much more seri-
ous effects that are likely to occur with continued or more intense lead exposure. Although
most commonly seen in industrial exposure cases, colic is also a lead-poisoning symptom pres-
ent in infants and young children.
Beritic (1971) examined 64 men suffering from abdominal colic due to lead intoxication
through occupational exposure. The diagnosis of lead colic was based on the occurrence of
severe attacks of spasmodic abdominal pain accompanied by constipation, abnormally high copro-
porphyrinuria, excessive basophilic stippling, reticulocytosis, and some degree of anemia (all
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clinical signs of lead poisoning). Thirteen of the 64 patients had blood lead levels of
40-80 ug/dl upon admission. However, the report did not indicate how recently the patients'
exposures had been terminated or provide other details of their exposure histories.
A more recent report by Dahlgren (1978) focused on the gastrointestinal symptoms of lead
smelter workers whose blood lead levels were determined within two weeks of the termination of
their work exposure. Of 34 workers with known lead exposure, 27 (79 percent) complained of
abdominal pain, abnormal bowel movements, and nausea. Fifteen of the 27 had abdominal pain
for more than 3 months after removal from the exposure to lead. The mean (± SD) blood lead
concentration for this group of 15 was 70 (± 4) ug/dl. There was, however, no correlation
between severity of symptoms and blood lead levels, as those experiencing stomach pain for
less than 3 months averaged 68 (± 9) ug/dl and the remaining 7 workers, reporting no pain at
all, averaged 76 (± 9) ug/dl.
Haenninen et al. (1979) assessed the incidence of gastrointestinal symptoms in 45 workers
whose blood lead levels had been regularly monitored throughout their exposure and had never
exceded 69 ug/dl- A significant association between gastrointestinal symptoms (particularly
epigastric pain) and blood lead level was reported. This association was more pronounced in
subjects whose maximal blood lead levels had reached 50-69 ug/dl, but was also noted in those
whose blood lead levels were below 50 ug/dl.
Other occupational studies have also suggested a relationship between lead exposure and
gastrointestinal symptoms (Lilis et al., 1977; Irwig et al., 1978a,b; Fischbein et al., 1979,
1980). For demonstrating such a relationship, however, the most useful measure of internal
exposure has not necessarily been blood lead concentrations. Fischbein et al. (1980) surveyed
a cross-section of New York City telephone cable splicers exposed to lead in the process of
soldering cables. Of the 90 workers evaluated, 19 (21 percent) reported gastrointestinal
symptoms related to lead colic. The difference between mean blood lead levels in those
reporting GI symptoms and those not reporting such symptoms (30 versus 27 ug/dl) was not sta-
tistically significant. However, mean zinc protoporphyrin concentrations (67 versus 52 ug/dl)
were significantly different (p <0.02)
Limited experimental work has been devoted to gastrointestinal function either in humans
(Lerza and Fierro, 1958; Mungo and Sessa, 1960) or animals (Mambeeva, 1963; Cory-Slechta
etal., 1980). A recent study of chronically lead-exposed rats by Walsh and Ryden (1984)
indicated that concentrations of lead sufficient to cause renal and hematological toxicity did
not appreciably affect gastrointestinal transit.
Although gastrointestinal symptoms of lead exposure are clinically evident in frank lead
intoxication and may even be present when blood lead levels approach the 30-80 ug/dl range,
there is currently insufficient information to establish a clear dose-effect relationship for
the general population at ambient exposure levels.
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12.9.4 The Endocrine System
Some evidence exists for other effects of lead on the endocrine system, at least at high
levels of lead exposure. Lead is thought, for example, to decrease thyroid function In man
and experimental animals. Porritt (1931) suggested that lead dissolved from lead pipes by
soft water was the cause of hypothyroidism in individuals living in southwest England. Later
Kremer and Frank (1955) reported the simultaneous occurrence of myxedema and plumbism in a
house painter. Monaenkova (1957) observed impaired concentration of 131I by thyroid glands in
10 of 41 patients with industrial plumbism. Subsequently, Zel'tser (1962) showed that HI vivo
131I uptake and thyroxine synthesis by rat thyroid were decreased by lead when doses of 2 and
5 percent lead acetate solution were administered. Robins et al. (1983) reported a clinical
study of 12 workers with blood lead levels of 44 (jg/dl or above. Seven of the workers showed
low serum thyroxine and estimated free thyroxine levels. In the same report, the authors also
presented results of a cross-sectional study of 47 workers in which both of these indices of
thyroid function were negatively related to blood lead levels. The effects were more pro-
nounced in black men than in white men. Refowitz (1984) reported that he was unable to corro-
borate the findings of Robins et al. (1983). However, his regression plots of similar thyroid
function indices consistently showed negative relationships with blood lead levels in 58 work-
ers. Although these results did not achieve statistical significance in Refowitz1s (1984)
analysis, they suggested a stronger negative relationship between thyroxine and blood lead
levels in white workers than did the data of Robins et al. (1983).
Uptake of 131I, sometimes decreased in men with lead poisoning, can be offset by treat-
ment with thyroid-stimulating hormone (TSH) (Sandstead et al., 1969; Sandstead, 1967). Lead
may act to depress thyroid function by inhibiting thiol groups or by displacing iodine in a
protein sulfonyl iodine carrier (Sandstead, 1967), and the results suggest that excessive lead
may act at both the pituitary and the thyroid gland itself to impair thyroid function. None
of these effects on the thyroid system, however, have been demonstrated to occur in humans at
blood lead levels below 30-40 pg/dl.
Sandstead et al. (1970a) studied the effects of lead intoxication on pituitary and adre-
nal function in man and found that it may produce clinically significant hypopituitarism in
some. The effects of lead on adrenal function were less consistent, but some of the patients
showed a decreased responsiveness to an inhibitor (metapyrone) of 11-beta-hydroxylation in the
synthesis of cortisol. This suggests a possible impact of lead on pituitary-adrenal hormone
functions. That excessive oral ingestion of lead may in fact result in pathological changes
in the pituitary-adrenal axis is also supported by other reports (Murashov, 1966; Pines, 1965)
of lead-induced decreased metapyrone responsiveness, a depressed pituitary reserve, and
decreased immunoreactive adrenocorticotrophic hormone (ACTH). These same events may also
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affect adrenal gland function inasmuch as decreased urinary excretion of 17-hydroxy-
corticosteroids was observed in these patients. Furthermore, suppression of responsiveness to
exogenous ACTH in the zona fasciculate of the adrenal cortex has been reported in lead-
poisoned subjects (Makotchenko, 1965), and impairment of the zona glomerulosa of the adrenal
cortex has also been suggested (Sandstead et al., 1970b). Once again, however, none of these
effects on adrenal hormone function have been shown to occur at blood lead levels as low as
30-40 ug/dl,
Other studies provide evidence suggestive of lead exposure effects on endocrine systems
controlling reproductive functions (see also Section 12.6). For example, evidence of abnormal
luteinizing hormone (LH) secretory dynamics was found in secondary lead smelter workers
(Braunstein et al., 1978). Reduced basal serum testosterone levels with normal basal LH
levels, but a diminished rise in LH following stimulation, indicated suppression of
hypothalamic-pituitary function. Testicular biopsies in two lead-poisoned workmen showed
peritubular fibrosis suggesting direct toxic effects of lead in the testes as well as effects
at the hypothalamic-pituitary level. Lancranjan et al. (1975) also reported lead-related
interference with male reproductive functions. Moderately increased lead absorption (blood
lead mean = 52.8 ug/dl) among a group of 150 workmen who had long-term exposure to lead in
varying degrees was said to result in gonadal impairment. The effects on the testes were
believed to be direct, however, in that tests for hypothalamic-pituitary influence were nega-
tive.
In regard to effects of lead on ovarian function in human females, Panova (1972) reported
a study of 140 women working in a printing plant for 1-2 months, where ambient air lead levels
were <7 ug/m3. Using a classification of various age groups (20-25, 26-35, and 36-40 yr) and
type of ovarian cycle (normal, anovular, and disturbed lutein phase), Panova claimed that sta-
tistically significant differences existed between the lead-exposed and control groups in the
age range 20-25 years. It should be noted that the report does not show the age distribution,
the level of significance, or the data on specificity of the method used for classification.
Also, Zielhuis and Wibowo (1976), in a critical review of the above study, concluded that the
design of the study and presentation of data were such that it was difficult to evaluate the
author's conclusion that chronic exposure to low air lead levels leads to disturbed ovarian
function. Moreover, no consideration was given to the dust levels of lead, an important fac-
tor in print shops. Unfortunately, little else besides the above report exists in the litera-
ture in regard to assessing lead effects on human ovarian function or other factors affecting
human female fertility. Studies offering firm data on maternal variables such as hormonal
state, which is known to affect the ability of the pregnant woman to carry the fetus full-
term, are also lacking, although certain studies do at least indicate that high-level lead
exposure induces stillbirths and abortions (see Section 12.6).
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An animal study by Petrusz et al. (1979) reports that orally administered lead can exert
effects on pituitary and serum gonadotropins, which may represent one mechanism by which lead
affects reproductive functions. The blood lead levels at which alterations in serum and pitu-
itary follicle stimulating hormone were observed in neonatal rats, however, were well in ex-
cess of 100 ug/dl. (Evidence relating endocrine function to various recently reported lead-
associated effects on human fetal and child development, including effects on growth and
stature, is reviewed in an Addendum to this document.)
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12.10 CHAPTER SUMMARY
12.10.1 Introduction
Lead has diverse biological effects in humans and animals. Its effects are seen at the
subcellular level of organellar structures and processes as well as at the overall level of
general functioning that encompasses all systems of the body operating in a coordinated,
interdependent fashion. The present chapter not only categorizes and describes the various
biological effects of lead but also attempts to identify the exposure levels at which such
effects occur and the mechanisms underlying them. The dose-response curve for the entire
range of biological effects exerted by lead is rather broad, with certain biochemical changes
occurring at relatively low levels of exposure and perturbations in other systems, such as the
liver, becoming detectable only at relatively high exposure levels. In terms of relative
vulnerability to deleterious effects of lead, the developing organism generally appears to be
more sensitive than the mature individual. Additional, quantitative examination of overall
exposure-effect relationships for lead is presented in Chapter 13. It should be noted that
lead has no known beneficial biological effects. Available evidence does not demonstrate that
lead is an essential element.
12.10.2 Subcellular Effects of Lead
The biological basis of lead toxicity is its ability to bind to ligating groups in bio-
molecular substances crucial to various physiological functions, thereby interfering with
these functions by, for example, competing with native essential metals for binding sites,
inhibiting enzyme activity, and inhibiting or otherwise altering essential ion transport.
These effects are modulated by the following: 1) the inherent stability of such binding sites
for lead; 2) the compartmentalization kinetics governing lead distribution among body compart-
ments, among tissues, and within cells; and 3) the differences in biochemical organization
across cells and tissues due to their specific functions. Given the complexities introduced
by items 2 and 3, it. is not surprising that no single unifying mechanism of lead toxicity
across all tissues in humans and experimental animals has yet been demonstrated.
Insofar as effects of lead on activity of various enzymes are concerned, many of the
available studies concern ijn vitro behavior of relatively pure enzymes with marginal relevance
to various effects rn vivo. On the other hand, certain enzymes are basic to the effects of
lead at the organ or organ system level, and discussion is best reserved for such effects in
the summary sections below dealing with lead's effects on particular organ systems. This sec-
tion is mainly concerned with organellar effects of lead, especially those which provide some
rationale for lead toxicity at higher levels of biological organization. Particular emphasis
is placed on the mitochondrion, because this organelle is not only affected by lead in numer-
ous ways but has also provided the most data bearing on the subcellular effects of lead.
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The critical target organelle for lead toxicity in a variety of cell and tissue types
clearly is the mitochondrion, followed probably by cellular and intracellular membranes. The
mitochondrial effects take the form of structural changes and marked disturbances in mitochon-
drial function within the cell, particularly in energy metabolism and ion transport. These
effects in turn are associated with demonstrable accumulation of lead in mitochondria, both 1n
vivo and jni vitro. Structural changes include mitochondrial swelling in a variety of ceTT
types as well as distortion and loss of cristae, which occur at relatively moderate lead
levels. Similar changes have also been documented in lead workers across a range of ex-
posures.
Uncoupled energy metabolism, inhibited cellular respiration using both succinate and
nicotinamide adenine dinucleotide (NAD)-linked substrates, and altered kinetics of intracellu-
lar calcium have been demonstrated ni vivo using mitochondria of brain and non-neural tissues
In some cases, the lead exposure level associated with such changes has been relatively low.
Several studies document the relatively greater sensitivity of this organelle in young versus
adult animals in terms of mitochondrial respiration. The cerebellum appears to be particular-
ly sensitive, providing a connection between mitochondrial impairment and lead encephalopathy.
Lead's impairment of mitochondrial function in the developing brain has also been consistently
associated with delayed brain development, as indexed by content of various cytochromes. jn
the rat pup, ongoing lead exposure from birth is required for this effect to be expressed,
indicating that such exposure must occur before, and is inhibitory to, the burst of oxidative
metabolism activity that occurs in the young rat at 10-21 days postnatally.
In vivo lead exposure of adult rats also markedly inhibits calcium turnover in a cellular
compartment of the cerebral cortex that appears to be the mitochondrion. This effect has been
seen at a brain lead level of 0.4 M9/9- These results are consistent with a separate study
showing increased retention of calcium in the brains of lead-dosed guinea pigs. Numerous re-
ports have described the HI V1V° accumulation of lead in mitochondria of kidney, liver
spleen, and brain tissue, with one study showing that such uptake was slightly more than
occurred in the cell nucleus. These data are not only consistent with deleterious effects of
lead on mitochondria but are also supported by other investigations w vitro. Significant
decreases in mitochondrial respiration jn vitro using both NAD-1inked and succinate substrates
have been observed for brain and non-neural tissue mitochondria in the presence of lead at
micromolar levels. There appears to be substrate specificity in the inhibition of respiration
across different tissues, which may be a factor in differential organ toxicity. Also, a
number of enzymes involved in intermediary metabolism in isolated mitochondria have been ob-
served to undergo significant inhibition of activity with lead.
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Of particular interest regarding lead's effects on isolated mitochondria are ion trans-
port effects, especially in regard to calcium. Lead movement into brain and other tissue
mitochondria involves active transport, as does calcium. Recent sophisticated kinetic
analyses of desaturation curves for radiolabeled lead or calcium indicate that there is
striking overlap in the cellular metabolism of calcium and lead. These studies not only
establish the basis for the easy entry of lead into cells and cell compartments, but also pro-
vide a basis for lead's impairment of intracellular ion transport, particularly in neural cell
mitochondria, where the capacity for calcium transport is 20-fold higher than even in heart
mitochondria.
Lead is also selectively taken up in isolated mitochondria ui vitro, including the mito-
chondria of synaptosomes and brain capillaries. Given the diverse and extensive evidence of
lead's impairment of mitochondria! structure and function as viewed from a subcellular level,
it is not surprising that these derangements are logically held to be the basis of dysfunction
of heme biosynthesis, erythropoiesis, and the central nervous system. Several key enzymes in
the heme biosynthetic pathway are intramitochondrial, particularly ferrochelatase. Hence, it
is to be expected that entry of lead into mitochondria will impair overall heme biosynthesis,
and in fact this appears to be the case in the developing cerebellum. Furthermore, rela-
tively moderate levels of lead may be associated with its entry into mitochondria and conse-
quent expressions of mitochondrial injury.
Lead exposure provokes a typical cellular reaction in humans and other species that has
been morphologically characterized as a lead-containing nuclear inclusion body. While it has
been postulated that such inclusions constitute a cellular protection mechanism, such a
mechanism is an imperfect one. Other organelles, e.g., the mitochondrion, also take up lead
and sustain injury in the presence of nuclear inclusion formations.
In theory, the cell membrane is the first organelle to encounter lead and it is not
surprising that cellular effects of lead can be ascribed to interactions at cellular and
intracellular membranes in the form of disturbed ion transport. The inhibition of membrane
(Na+,K+)-ATPase of erythrocytes as a factor in lead-impaired erythropoiesis is noted else-
where. Lead also appears to interfere with the normal processes of calcium transport across
membranes of different tissues. In peripheral cholinergic synaptosomes, lead is associated
with retarded release of acetylcholine owing to a blockade of calcium binding to the membrane,
while calcium accumulation within nerve endings can be ascribed to inhibition of membrane
(Na+,K+)-ATPase.
Lysosomes accumulate in renal proximal convoluted tubule cells of rats and rabbits given
lead over a range of dosing. This also appears to occur in the kidneys of lead workers and
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seems to represent a disturbance in normal lysosomal function, with the accumulation of lyso-
somes being due to enhanced degradation of proteins because of the effects of lead elsewhere
within the cell.
12.10.3. Effects of Lead on Heme Biosynthesis, Erythropoiesis, and Erythrocyte Physiology in
Humans and Animals'
The effects of lead on heme biosynthesis are well known because of their clinical promi-
nence and the numerous studies of such effects in humans and experimental animals. The pro-
cess of heme biosynthesis starts with glycine and succinyl-coenzyme A, proceeds through forma-
tion of protoporphyrin IX, and culminates with the insertion of divalent iron into the por-
phyrin ring to form heme. In addition to being a constituent of hemoglobin, heme is the
prosthetic group of many tissue hemoproteins having variable functions, such as myoglobin, the
P-450 component of the mixed-function oxygenase system, and the cytochromes of cellular ener-
getics. Hence, disturbance of heme biosynthesis by lead poses the potential for multiple-
organ toxicity.
In investigations of lead's effects on the heme synthesis pathway, most attention has
been devoted to the following: (1) stimulation of mitochondrial delta-aminolevulinic acid
synthetase (ALA-S), which mediates formation of delta-aminolevulinic acid (ALA); (2) direct
inhibition of the cytosolic enzyme, delta-aminolevulinic acid dehydrase (ALA-D), which cata-
lyzes formation of porphobilinogen from two units of ALA; and (3) inhibition of insertion of
iron (II) into protoporphyrin IX to form heme, a process mediated by ferrochelatase.
Increased ALA-S activity has been found in lead workers as well as in lead-exposed ani-
mals, although an actual decrease in enzyme activity has also been observed in several experi-
mental studies using different exposure methods. It appears, then, that the effect on ALA-s
activity may depend on the nature of the exposure. Using rat liver cells in culture, ALA-S
activity was stimulated jji vitro at lead levels as low as 5.0 uM or 1.0 ug/g preparation. The
increased activity was due to biosynthesis of more enzyme. The blood lead threshold for stim-
ulation of ALA-S activity in humans, based on a study using leukocytes from lead workers,
appears to be about 40 pg/dl. Whether this apparent threshold applies to other tissues
depends on how well the sensitivity of leukocyte mitochondria mirrors that in other systems.
The relative impact of ALA-S activity stimulation on ALA accumulation at lower lead exposure
levels appears to be much less than the effect of ALA-D activity inhibition. ALA-D activity
is significantly depressed at 40 ug/dl blood lead, the point at which ALA-S activity only
begins to be affected.
Erythrocyte ALA-D activity is very sensitive to inhibition by lead. This inhibition is
reversed by reactivation of the sulfhydryl group with agents such as dithiothreitol, zinc, or
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zinc and glutathione. Zinc levels that achieve reactivation, however, are well above physio-
logical levels. Although zinc appears to offset the inhibitory effects of lead observed in
animal studies and in human erythrocytes rn vitro, lead workers exposed to both zinc and lead
do not show significant changes in the relationship of ALA-D activity to blood lead when com-
pared with workers exposed just to lead. Nor does the range of physiological zinc levels in
nonexposed subjects affect ALA-D activity. In contrast, zinc deficiency in animals signifi-
cantly inhibits ALA-D activity, with concomitant accumulation of ALA in urine. Because zinc
deficiency has also been demonstrated to increase lead absorption, the possibility exists for
the following dual effects of such deficiency on ALA-D activity: (1) a direct effect on acti-
vity due to reduced zinc availability; and (2) increased lead absorption leading to further
inhibition of activity.
Erythrocyte ALA-D activity appears to be inhibited at virtually all blood lead levels
measured so far, and any threshold for this effect in either adults or children remains to be
determined. A further measure of this enzyme's sensitivity to lead is a report that rat bone
marrow suspensions show inhibition of ALA-D activity by lead at a level of 0.1 ug/g suspen-
sion. Inhibition of ALA-D activity in erythrocytes apparently reflects a similar effect in
other tissues. Hepatic ALA-D activity in lead workers was inversely correlated with erythro-
cyte activity as well as blood lead levels. Of significance are experimental animal data
showing that (1) brain ALA-D activity is inhibited with lead exposure, and (2) this inhibition
appears to occur to a greater extent in developing animals than in adults, presumably reflec-
ting greater retention of lead in developing animals. In the avian brain, cerebellar ALA-D
activity is affected to a greater extent than that of the cerebrum and, relative to lead con-
centration, shows inhibition approaching that occurring in erythrocytes.
Inhibition of ALA-D activity by lead is reflected by elevated levels of its substrate,
ALA, in blood, urine, and soft tissues. Urinary ALA is employed extensively as an indicator
of excessive lead exposure in lead workers. The diagnostic value of this measurement in pedi-
atric screening, however, is limited when only spot urine collection is done; more satisfac-
tory data are obtainable with 24-hr collections. Numerous independent studies document a
direct correlation between blood lead and the logarithm of urinary ALA in human adults and
children; the blood lead threshold for increases in urinary ALA is commonly accepted as 40
ug/dl. However, several studies of lead workers indicate that the correlation between urinary
ALA and blood lead continues below this value; one study found that the slope of the dose-
effect curve in lead workers depends on the level of exposure.
The health significance of lead-inhibited ALA-D activity and accumulation of ALA at lower
lead exposure levels is controversial. The "reserve capacity" of ALA-D activity is such that
only the level of inhibition associated with marked accumulation of the enzyme's substrate,
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ALA, in accessible indicator media may be significant. However, it is not possible to quan-
tify at lower levels of lead exposure the relationship of urinary ALA to target tissue levels
or to relate the potential neurotoxicity of ALA at any accumulation level to levels in indi-
cator media. Thus, the blood lead threshold for neurotoxicity of ALA may be different from
that associated with increased urinary excretion of ALA.
Accumulation of protoporphyrin in erythrocytes of lead-intoxicated individuals has been
recognized since the 1930s, but it has only recently been possible to quantitatively assess
the nature of this effect via development of sensitive, specific microanalysis methods. Accu-
mulation of protoporphyrin IX in erythrocytes results from impaired placement of iron (II) in
the porphyrin moiety in heme formation, an intramitochondrial process mediated by ferrochela-
tase. In lead exposure, the porphyrin acquires a zinc ion in lieu of native iron, thus form-
ing zinc protoporphyrin (2PP), which is tightly bound in available heme pockets for the life
of the erythrocytes. This tight sequestration contrasts with the relatively mobile nonmetal,
or free, erythrocyte protoporphyrin (FEP) accumulated in the congenital disorder erythropoiet-
ic protoporphyria.
Elevation of erythrocyte ZPP has been extensively documented as exponentially correlated
with blood lead in children and adult lead workers and is currently considered one of the best
indicators of undue lead exposure. Accumulation of ZPP only occurs in erythrocytes formed
during lead's presence in erythroid tissue; this results in a lag of at least several weeks
before its buildup can be measured. The level of ZPP accumulation in erythrocytes of newly
employed lead workers continues to increase after blood lead has already reached a plateau.
This influences the relative correlation of ZPP and blood lead in workers with short exposure
histories. Also, the ZPP level in blood declines much more slowly than blood lead, even after
removal from exposure or after a drop in blood lead. Hence, ZPP level appears to be a more
reliable indicator of continuing intoxication from lead resorbed from bone.
The threshold for detection of lead-induced ZPP accumulation is affected by the relative
spread of blood lead and corresponding ZPP values measured. In young children (<4 yr old),
the ZPP elevation associated with iron-deficiency anemia must also be considered. In adults,
numerous studies indicate that the blood lead threshold for ZPP elevation is about 25-30
ug/dl. In children 10-15 years old, the threshold is about 16 ug/dl; for this age group, iron
deficiency is not a factor. In one study, children over 4 years old showed the same thresh-
old, 15.5 ug/dl, as a second group under 4 years old, indicating that iron deficiency was not
a factor in the study. At 25 ug/dl blood lead, 50 percent of the children had significantly
elevated FEP levels (2 standard deviations above the reference mean FEP).
At blood lead levels below 30-40 ug/dl, any assessment of the EP-blood lead relationship
is strongly influenced by the relative analytical proficiency of measurements of both blood
lead and EP. The types of statistical analyses used are also important. In a recent detailed
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statistical study involving 2004 children, 1852 of whom had blood lead values below 30 ug/dl,
segmental line and probit analysis techniques were employed to assess the dose-effect thres-
hold and dose-response relationship. An average blood lead threshold for the effect using
both statistical techniques was 16.5 ug/dl for the full group and for those subjects with
blood lead below 30 ug/dl. The effect of iron deficiency was tested for and removed. Of par-
ticular interest was the finding that blood lead values of 28.6 and 35.7 ug/dl corresponded to
EP elevations more than 1 or 2 standard deviations, respectively, above the reference mean in
50 percent of the children. Hence, fully half of the children had significant elevations of
EP at blood lead levels around 30 |jg/dl. From various reports, children and adult females
appear to be more sensitive to lead's effects on EP accumulation at any given blood lead
level; children are somewhat more sensitive than adult females.
Lead's effects on heme formation are not restricted to the erythropoietic system. Recent
studies show that the reduction of serum 1,25-dihydroxyvitamin D seen with even low-level lead
exposure is apparently the result of lead-induced inhibition of the activity of renal
1-hydroxylase, a cytochrome P-450 mediated enzyme. Reduction in activity of the hepatic
enzyme tryptophan pyrrolase and concomitant increases in plasma tryptophan as well as brain
tryptophan, serotonin, and hydroxyindoleacetic acid have been shown to be associated with
lead-induced reduction of the hepatic heme pool. The heme-containing protein cytochrome P-450
(an integral part of the hepatic mixed-function oxygenase system) is affected in humans and
animals by lead exposure, especially acute intoxication. Reduced P-450 content correlates
with impaired activity of detoxifying enzyme systems such as aniline hydroxylase and aminopy-
rine demethylase. It is also responsible for reduced 6p-hydroxylation of cortisol in children
having moderate lead exposure.
Studies of organotypic chick and mouse dorsal root ganglion in culture show that the ner-
vous system has heme biosynthetic capability and that not only is such capability reduced in
the presence of lead but production of porphyrinic material is increased. In the neonatal
rat, chronic lead exposure resulting in moderately elevated blood lead is associated with
retarded increases in the hemoprotein cytochrome C and with disturbed electron transport in
the developing cerebral cortex. These data parallel effects of lead on ALA-D activity and ALA
accumulation in neural tissue. When both of these effects are viewed in the toxicokinetic
context of increased retention of lead in both developing animals and children, there is an
obvious and serious potential for impaired heme-based metabolic function in the nervous system
of lead-exposed children.
As can be concluded from the above discussion, the health significance of ZPP accumula-
tion rests with the fact that it is evidence of impaired heme and hemoprotein formation in
many tissues that arises from entry of lead into mitochondria. Such evidence for reduced heme
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synthesis is consistent with a great deal of data documenting lead-associated effects on mito-
chondria. The relative value of the lead-ZPP relationship in erythropoietic tissue as an
index of this effect in other tissues hinges on the relative sensitivity of the erythropoietic
system compared with other organ systems. One study of rats exposed over their lifetime to
low levels of lead demonstrated that protoporphyrin accumulation in renal tissue was already
significant at levels of lead exposure which produced little change in erythrocyte porphyrin
levels.
Other steps in the heme biosynthesis pathway are also known to be affected by lead, al-
though these have not been as well studied on a biochemical or molecular level. Coproporphy-
rin levels are increased in urine, reflecting active lead intoxication. Lead also affects the
activity of the enzyme uroporphyrinogen-I-synthetase in experimental animal systems, resulting
in an accumulation of its substrate, porphobilinogen. The erythrocyte enzyme has been report-
ed to be much more sensitive to lead than the hepatic species, presumably accounting for much
of the accumulated substrate. Unlike the case with experimental animals, lead-exposed humans
show no rise in urinary porphobilinogen, which is a differentiating characteristic of lead
intoxication versus the hepatic porphyrias. Ferrochelatase is an intramitochondrial enzyme
and impairment of its activity, either directly by lead or via impairment of iron transport to
the enzyme, is evidence of the presence of lead in mitochondria.
Anemia is a manifestation of chronic lead intoxication and is characterized as mildly
hypochromic and usually normocytic. It is associated with reticulocytosis, owing to shortened
cell survival, and the variable presence of basophilic stippling. Its occurrence is due to
both decreased production and increased rate of destruction of erythrocytes. In young chil-
dren (<4 yr old), iron deficiency anemia is exacerbated by lead uptake, and vice versa. Hemo-
globin production is negatively correlated with blood lead in young children, in whom iron
deficiency may be a confounding factor, as well as in lead workers. In one study, blood lead
values that were usually below 80 ug/dl were inversely correlated with hemoglobin content. In
these subjects no iron deficiency was found. The blood lead threshold for reduced hemoglobin
content is about 50 ug/dl in adults and somewhat lower (~40 pg/dl) in children.
The mechanism of lead-associated anemia appears to be a combination of reduced hemoglobin
production and shortened erythrocyte survival due to direct cell injury. Lead's effects on
hemoglobin production involve disturbances of both heme and globin biosynthesis. The hemoly-
tic component to lead-induced anemia appears to be caused by increased cell fragility and in-
creased osmotic resistance. In one study using rats, the hemolysis associated with vitamin E
deficiency, via reduced cell deformability, was exacerbated by lead exposure. The molecular
basis for increased cell destruction rests with inhibition of (Na+, K+)-ATPase and pyrimidine-
S'-nucleotidase. Inhibition of the former enzyme leads to cell "shrinkage" and inhibition of
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the latter results in impaired pyrimidine nucleotide phosphorolysis and disturbance of the
activity of the purine nuclcotides necessary for cellular energetics.
In lead intoxication, the presence of both basophilic stippling and anemia with a hemo-
lytic component is due to inhibition by lead of the activity of pyrimidine-S'-nucleotidase
(Py-5-N), an enzyme that mediates the dephosphorylation of pyrimidine nucleotides in the
maturing erythrocyte. Inhibition of this enzyme by lead has been documented in lead workers,
lead-exposed children, and experimental animal models. In one study of lead-exposed children,
there was a negative correlation between blood lead and enzyme activity, with no clear re-
sponse threshold. A related report noted that, in addition, there was a positive correlation
between cytidine phosphate and blood lead and an inverse correlation between pyrimidine
nucleotide and enzyme activity.
The metabolic significance of Py-5-N inhibition and cell nucleotide accumulation is that
they affect erythrocyte stability and survival as well as potentially affect mRNA and protein
synthesis related to globin chain synthesis. Based on one study of children, the threshold
for the inhibition of Py-5-N activity appears to be about 10 ug/dl blood lead. Lead's inhibi-
tion of Py-5-N activity and a threshold for such inhibition are not by themselves the issue.
Rather, the issue is the relationship of such inhibition to a significant level of impaired
pyrimidine nucleotide metabolism and the consequences for erythrocyte stability and function.
The relationship of Py-5-N activity inhibition by lead to accumulation of its pyrimidine
nucleotide substrate is analogous to lead's inhibition of ALA-D activity and accumulation of
ALA.
Tetraethyl lead and tetramethyl lead, components of leaded gasoline, undergo transforma-
tion KJ vivo to neurotoxic trialkyl metabolites as well as further conversion to inorganic
lead. Hence, one might anticipate that exposure, to such agents may result in effects commonly
associated with inorganic lead, particularly in terms of heme synthesis and erythropoiesis.
Various surveys and case reports show that the habit of sniffing leaded gasoline is associated
with chronic lead intoxication in children from socially deprived backgrounds in rural or re-
mote areas. Notable in these subjects is evidence of impaired heme biosynthesis, as indexed
by significantly reduced ALA-D activity. In several case reports of frank lead toxicity from
habitual leaded gasoline sniffing, effects such as basophilic stippling in erythrocytes and
significantly reduced hemoglobin have also been noted.
The role of lead-associated disturbances of heme biosynthesis as a possible factor in
neurological effects of lead is of considerable interest due to the following: (1) simi-
larities between classical signs of lead neurotoxicity and several neurological components of
the congenital disorder acute intermittent porphyria; and (2) some of the unusual aspects of
lead neurotoxicity. There are three possible points of connection between lead's effects on
heme biosynthesis and the nervous system. Associated with both lead neurotoxicity and acute
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intermittent porphyria is the common feature of excessive systemic accumulation and excretion
of ALA. In addition, lead neurotoxicity reflects, to some degree, impaired synthesis of heme
and hemoproteins involved in crucial cellular functions; such an effect on heme is now known
to be relevant within neural tissue as well as in non-neural tissue.
Available information indicates that ALA levels are elevated in the brains of lead-
exposed animals and arise through in situ inhibition of brain ALA-D activity or through trans-
port of ALA to the brain after formation in other tissues. ALA is known to traverse the
blood-brain barrier. Hence, ALA is accessible to, or formed within, the brain during lead
exposure and may express its neurotoxic potential.
Based on various jn vitro and HI vivo neurochemical studies of lead neurotoxicity, it
appears that ALA can inhibit release of the neurotransmitter gamma-aminobutyric acid (GABA)
from presynaptic receptors at which ALA appears to be very potent even at low levels. In an
jin vitro study, ALA acted as an agonist at levels as low as 1.0 uM ALA. This jn vitro obser-
vation supports results of a study using lead-exposed rats in which there was inhibition of
both resting and K -stimulated release of preloaded 3H-GABA from nerve terminals. The obser-
vation that jji vivo effects of lead on neurotransmitter function cannot be duplicated with in
vitro preparations containing added lead is further evidence of an effect of some agent (other
than lead) that acts directly on this function. Human data on lead-induced associations
between disturbed heme synthesis and neurotoxicity, while limited, also suggest that ALA may
function as a neurotoxicant.
A number of studies strongly suggest that lead-impaired heme production itself may be a
factor in the toxicant's neurotoxicity. In porphyric rats, lead inhibits tryptophan pyrrolase
activity owing to reductions in the hepatic heme pool, thereby leading to elevated levels of
tryptophan and serotonin in the brain. Such elevations are known to induce many of the neuro-
toxic effects also seen with lead exposure. Of great interest is the fact that heme infusion
in these animals reduces brain levels of these substances and also restores enzyme activity
and the hepatic heme pool. Another line of evidence for the heme-basis of lead neurotoxicity
is that mouse dorsal root ganglion in culture manifests morphological evidence of neural in-
jury with rather low lead exposure, but such changes are largely prevented with co-admini-
stration of heme. Finally, studies also show that heme-requiring cytochrome C production is
impaired along with operation of the cytochrome C respiratory chain in the brain when neonatal
rats are exposed to lead.
Awareness of the interactions of lead and the vitamin D-endocrine system has been grow-
ing. A recent study has found that children with blood lead levels of 33-120 ug/dl showed
significant reductions in serum levels of the hormonal metabolite 1,25-dihydroxyvitamin D
(1,25~(OH)2D). This inverse dose-response relationship was found throughout the range of
measured blood lead values, 12-120 ug/dl, and appeared to be the result of lead's effect on
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the production of the vitamin D hormone. The 1,25-(OH)2D levels of children with blood lead
levels of 33-55 ug/dl corresponded to the levels that have been observed in children with
severe renal dysfunction. At higher blood lead levels (>62 ug/dl), the 1,25-(OH)2D values
were similar to those that have been measured in children with various inborn metabolic dis-
orders. Chelation therapy of the lead-poisoned children (blood lead levels >62 pg/dl) resulted
in a return to normal 1,25-(OH)2D levels within a short period.
In addition to its well-known actions on bone remodeling and intestinal absorption of
minerals, the vitamin D hormone has several other physiological actions at the cellular level.
These include cellular calcium homeostasis in virtually all mammalian cells and associated
calcium-mediated processes that are essential for cellular integrity and function. In addi-
tion, the vitamin D hormone has newly recognized functions that involve cell differentiation
and essential immunoregulatory capacity. It is reasonable to conclude, therefore, that im-
paired production of 1,25-(OH)2D can have profound and pervasive effects on tissues and cells
of diverse type and function throughout the body.
12.10.4 Neurotoxic Effects of Lead
An assessment of the impact of lead on human and animal neurobehavioral function raises a
number of issues. Among the key points addressed here are the following: (1) the internal
exposure levels, as indexed by blood lead levels, at which various neurotoxic effects occur;
(2) the persistence or reversibility of such effects; and (3) populations that appear to be
most susceptible to neural damage. In addition, the question arises as to the utility of
using animal studies to draw parallels to the human condition.
12.10.4.1 Internal Lead Levels at which Neurotoxic Effects Occur. Markedly elevated blood
lead levels are associated with the most serious neurotoxic effects of lead exposure (includ-
ing severe, irreversible brain damage as indexed by the occurrence of acute or chronic enceph-
alopathic symptoms, or both) in both humans and animals. For most adult humans, such damage
typically does not occur until blood lead levels exceed 120 ug/dl. Evidence does exist, how-
ever, for acute encephalopathy and death occurring in some human adults at blood lead as low
as 100 ug/dl. In children, the effective blood lead level for producing encephalopathy or
death is lower, starting at approximately 80-100 ug/dl. It should be emphasized that, once
encephalopathy occurs, death is not an improbable outcome, regardless of the quality of medi-
cal treatment available at the time of acute crisis. In fact, certain diagnostic or treatment
procedures themselves may exacerbate matters and push the outcome toward fatality if the
nature and severity of the problem are not diagnosed or fully recognized. It is also crucial
to note the rapidity with which acute encephalopathic symptoms can develop or death can occur
in apparently asymptomatic individuals or in those apparently only mildly affected by elevated
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lead body burdens. Rapid deterioration often occurs, with convulsions or coma suddenly ap-
pearing with progression to death within 48 hours. This strongly suggests that even in
apparently asymptomatic individuals, rather severe neural damage probably exists at high blood
lead levels even though it is not yet overtly manifested in obvious encephalopathic symptoms.
This conclusion is further supported by numerous studies showing that overtly lead intoxicated
children with high blood lead levels, but not observed to manifest acute encephalopathic symp-
toms, are permanently cognitively impaired, as are most children who survive acute episodes of
frank lead encephalopathy.
Recent studies show that overt signs and symptoms of neurotoxicity (indicative of both
CNS and peripheral nerve dysfunction) are detectable in some human adults at blood lead levels
as low as 40-60 ug/dl, levels well below blood lead concentrations previously thought to be
"safe" for adult lead exposures. In addition, certain electrophysiological studies of peri-
pheral nerve function in lead workers indicate that slowing of nerve conduction velocities in
some peripheral nerves is associated with blood lead levels as low as 30-50 ug/dl (with no
clear threshold for the effect being evident). These results are indicative of neurological
dysfunctions occurring at relatively low lead levels in non-overtly lead-intoxicated adults.
Other evidence confirms that neural dysfunctions exist in apparently asymptomatic chil-
dren at similar or even lower levels of blood lead. The body of studies on low- or moderate-
level lead effects on neurobehavioral functions in non-overtly lead-intoxicated children, as
summarized in Table 12-2, presents an array of data pointing to that conclusion. At high
exposure levels, several studies point toward average 5-point IQ decrements occurring in
asymptomatic children at average blood levels of 50-70 ug/dl. Other evidence is indicative of
average IQ decrements of about 4 points being associated with blood levels in a 30-50 ug/dl
range. Below 30 ug/dl, the evidence for IQ decrements is quite mixed, with some studies show-
ing no significant associations with lead once other confounding factors are controlled.
Still, the 1-2 point differences in IQ generally seen with blood lead levels in the 15-30
ug/dl range are suggestive of lead effects that are often dwarfed by other socio-hereditary
factors. Moreover, a highly significant linear relationship between IQ and blood lead over
the range of 6 to 47 ug/dl found in low-SES Black children indicates that IQ effects may be
detected without evident threshold even at these low levels, at least in this population of
children. In addition, other behavioral (e.g., reaction time, psychomotor performance) and
electrophysiological (altered EEG patterns, evoked potential measures, and peripheral nerve
conduction velocities) are consistent with a dose-response function relating neurotoxic ef-
fects to lead exposure levels as low as 15-30 ug/dl and possibly lower. Although the com-
parability of blood lead concentrations across species is uncertain (see discussion below),
studies show neurobehavioral effects in rats and monkeys at maximal blood lead levels below
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20 ug/dl; some studies demonstrate residual effects long after lead exposure has terminated
and blood lead levels have returned to approximately normal levels.
Timing, type, and duration of exposure are important factors in both animal and human
studies. It is often uncertain whether observed blood lead levels represent the levels that
were responsible for observed behavioral deficits or electrophysiological changes. Monitoring
of lead exposures in pediatric subjects in all cases has been highly intermittent or non-
existent during the period of life preceding neurobehavioral assessment. In most studies of
children, only one or two blood lead values are provided per subject. Tooth lead may be an
important cumulative exposure index, but its modest, highly variable correlation to blood
lead, FEP, or external exposure levels makes findings from various studies difficult to com-
pare quantitatively. The complexity of the many important covariates and their interaction
with dependent variable measures of modest validity, e.g., IQ tests, may also account for many
of the discrepancies among the different studies.
12.10.4.2 Jhe Question of Irreversibility. Little research on humans is available on persis-
tence of effects. Some work suggests that mild forms of peripheral neuropathy in lead workers
may be reversible after termination of lead exposure, but little is known regarding the rever-
sibility of lead effects on central nervous system function in humans. A two-year follow-up
study of 28 children of battery factory workers found a continuing relationship between blood
lead levels and altered slow wave voltage of cortical slow wave potentials indicative of per-
sisting CNS effects of lead, and a five-year follow-up of some of the same children revealed
the presence of altered brain stem auditory evoked potentials. Current population studies,
however, will have to be supplemented by longitudinal studies of the effects of lead on
development in order to address the issue of the reversibility or persistence of the neuro-
toxic effects of lead in humans more satisfactorily. (See the Addendum to this document for
a discussion of recent results from prospective studies linking perinatal lead exposure to
postnatal mental development.)
Various animal studies provide evidence that alterations in neurobehavioral function may
be long-lived, with such alterations being evident long after blood lead levels have returned
to control levels. These persistent effects have been demonstrated in monkeys as well as rats
under a variety of learning performance test paradigms. Such results are also consistent with
morphological, electrophysiological, and biochemical studies on animals that suggest lasting
changes in synaptogenesis, dendritic development, myelin and fiber tract formation, ionic
mechanisms of neurotransmission, and energy metabolism.
12.10.4.3 Early Development and the Susceptibility to Neural Damage. On the question of
early childhood vulnerability, the neurobehavioral data are consistent with morphological and
biochemical studies of the susceptibility of the heme biosynthetic pathway to perturbation by
lead. Various lines of evidence suggest that the order of susceptibility to lead's effects is
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as follows: (1) young > adults and (2) female > male. Animal studies also have pointed to
the perinatal period of ontogeny as a particularly critical time for a variety of reasons:
(1) it is a period of rapid development of the nervous system; (2) it is a period when good
nutrition is particularly critical; and (3) it is a period when the caregiver environment is
vital to normal development. However, the precise boundaries of a critical period are not yet
clear and may vary depending on the species and function or endpoint that is being assessed.
One analysis of lead-exposed children suggests that differing effects on cognitive performance
may be a function of the different ages at which children are subjected to neurotoxic expo-
sures. Nevertheless, there is general agreement that human infants and toddlers below the age
of three years are at special risk because of jji utero exposure (see Addendum), increased
opportunity for exposure because of normal mouthing behavior, and increased rates of lead
absorption due to various factors, e.g., nutritional deficiences.
12.10.4.4 Utility of Animal Studies in Drawing Parallels to the Human Condition. Animal
models are used to shed light on questions where it is impractical or ethically unacceptable
to use human subjects. This is particularly true in the case of exposure to environmental
toxins such as lead. In the case of lead, it has been effective and convenient to expose
developing animals via their mothers' milk or by gastric gavage, at least until weaning. In
many studies, exposure was continued in the water or food for some time beyond weaning. This
approach simulates at least two features commonly found in human exposure: oral intake and
exposure during early development. The preweaning period in rats and mice is of particular
relevance in terms of parallels with the first two years or so of human brain development.
Studies using rodents and monkeys have provided a variety of evidence of neurobehavioral
alteration induced by lead exposure. In most cases these effects suggest impairment in
"learning," i.e., the process of appropriately modifying one's behavior in response to infor-
mation from the environment. Such behavior involves the ability to receive, process, and
remember information in various forms. Some studies indicate behavioral alterations of a more
basic type, such as delayed development of certain reflexes. Other evidence suggests changes
affecting rather complex behavior in the form of social interactions.
Most of the above effects are evident in rodents and monkeys with blood lead levels ex-
ceeding 30 (jg/dl > but some effects on learning ability are apparent even at maximum blood lead
exposure levels below 20 ug/dl. Can these results with animals be generalized to humans?
Given differences between humans, rats, and monkeys in heme chemistry, metabolism, and other
aspects of physiology and anatomy, it is difficult to state what constitutes an equivalent
internal exposure level (much less an equivalent external exposure level). For example, is a
blood lead level of 30 ug/dl in a suckling rat equivalent to 30 ug/dl in a three-year-old
child? Until an answer is available for this question, i.e., until the function describing
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the relationship of exposure indices in different species is available, the utility of animal
models for deriving dose-response functions relevant to humans will be limited.
Questions also exist regarding the comparability of neurobehavioral effects in animals
with human behavior and cognitive function. One difficulty in comparing behavioral endpoints
such as locomotor activity is the lack of a consistent operational definition. In addition to
the lack of standardized methodologies, behavior is notoriously difficult to "equate" or com-
pare meaningfully across species because behavioral analogies do not demonstrate behavioral
homologies. Thus, it is improper to assume, without knowing more about the responsible under-
lying neurological structures and processes, that a rat's performance on an operant condition-
ing schedule or a monkey's performance on a stimulus discrimination task corresponds to a
child's performance on a cognitive function test. Nevertheless, interesting parallels in
hyper-reactivity and increased response variability do exist between different species, and
deficits in performance on various tasks are indicative of altered CMS functions, which are
likely to parallel some type of altered CNS function in humans as well.
In terms of morphological findings, there are reports of hippocampal lesions in both
lead-exposed rats and humans that are consistent with a number of behavioral findings suggest-
ing an impaired ability to respond appropriately to altered contingencies for rewards. That
is, subjects tend to persist in certain patterns of behavior even when changed conditions make
the behavior inappropriate. Other morphological findings in animals, such as demyelination
and glial cell decline, are comparable to human neuropathologic observations mainly at rela-
tively high exposure levels.
Another neurobehavioral endpoint of interest in comparing human and animal neurotoxicity
of lead is electrophysiological function. Alterations of electroencephalographic patterns and
cortical slow wave voltage have been reported for lead-exposed children, and various electro-
physiological alterations both i_n vivo (e.g., in rat visual evoked response) and JQ vitro
(e.g., in frog miniature endplate potentials) have also been noted in laboratory animals. At
this time, however, these lines of work have not converged sufficiently to allow for strong
conclusions regarding the electrophysiological aspects of lead neurotoxicity.
Biochemical approaches to the experimental study of lead's effects on the nervous system
have generally been limited to laboratory animal subjects. Although their linkage to human
neurobehavioral function is at this point somewhat speculative, such studies do provide in-
sight to possible neurochemical intermediaries of lead neurotoxicity. No single neurotrans-
mitter system has been shown to be particularly sensitive to the effects of lead exposure;
lead-induced alterations have been demonstrated in various neurotransmitters, including dopa-
mine, norepinephrine, serotonin, and y-aminobutyric acjd. In addition, lead has been shown to
have subcellular effects in the central nervous system at the level of mitochondrial function
and protein synthesis.
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Given the above-noted difficulties in formulating a comparative basis for internal expo-
sure levels among different species, the primary value of many animal studies, particularly in
vitro studies, may be in the information they can provide on basic mechanisms involved in lead
neurotoxicity. A number of jm vitro studies show that significant, potentially deleterious
effects on nervous system function occur at j_n situ lead concentrations of 5 pM and possibly
lower, suggesting that no threshold may exist for certain neurochemical effects of lead on a
subcellular or molecular level. The relationship between blood lead levels and lead concen-
trations at such extra- or intracellular sites of action, however, remains to be determined.
Despite the problems in generalizing from animals to humans, both the animal and the human
studies show great internal consistency in that they support a continuous dose-response func-
tional relationship between lead and neurotoxic biochemical, morphological, electrophysio-
logical, and behavioral effects.
12.10.5 Effects of Lead on the Kidney
It has been known for more than a century that kidney disease can result from lead
poisoning. Identifying the contributing causes and mechanisms of lead-induced nephropathy has
been difficult, however, in part because of the complexities of human exposure to lead and
other nephrotoxic agents. Nevertheless, it is possible to estimate at least roughly the range
of lead exposure associated with detectable renal dysfunction in both human adults and chil-
dren. Numerous studies of occupationally exposed workers have provided evidence for lead-
induced chronic nephropathy being associated with blood lead levels ranging from 40 to more
than 100 M9/dl» and some are suggestive of renal effects possibly occurring even at levels as
low as 30 ug/dl. In children, the relatively sparse evidence available points to the manifes-
tation of nephropathy only at quite high blood lead levels (usually exceeding 100-120 ug/dl).
The current lack of evidence for nephropathy at lower blood lead levels in children may simply
reflect the greater clinical concern with neurotoxic effects of lead intoxication in children
or, possibly, that much longer-term lead exposures are necessary to induce nephropathy. The
persistence of lead-induced nephropathy in children also remains to be more fully investi-
gated, although a few studies indicate that children diagnosed as being acutely lead poisoned
experience lead nephropathy effects lasting throughout adulthood.
Parallel results from experimental animal studies reinforce the findings in humans and
help illuminate the mechanisms underlying such effects. For example, a number of transient
effects in human and animal renal function are consistent with experimental findings of re-
versible lesions such as nuclear inclusion bodies, cytomegaly, swollen mitochondria, and
increased numbers of iron-containing lysosomes in proximal tubule cells. Irreversible lesions
such as interstitial fibrosis are also well documented in both humans and animals following
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chronic exposure to high doses of lead. Functional renal changes observed in humans have also
been confirmed in animal model systems with respect to increased excretion of amino acids and
elevated serum urea nitrogen and uric acid concentrations. The inhibitory effects of lead
exposure on renal blood flow and glomerular filtration rate are currently less clear in exper-
imental model systems; further research is needed to clarify the effects of lead on these
functional parameters in animals. Similarly, while lead-induced perturbation of the renin-
angiotensin system has been demonstrated in experimental animal models, further research is
needed to clarify the exact relationships among lead exposure (particularly chronic low-level
exposure), alteration of the renin-angiotensin system, and hypertension in both humans and
animals.
On the biochemical level, it appears that lead exposure produces changes at a number of
sites. Inhibition of membrane marker enzymes, decreased mitochondria! respiratory function/
cellular energy production, inhibition of renal heme biosynthesis, and altered nucleic acid
synthesis are the most marked changes to have been reported. The extent to which these mito-
chondrial alterations occur is probably mediated in part by the intracellular bioavailability
of lead, which is determined by its binding to high-affinity kidney cytosolic proteins and
deposition within intranuclear inclusion bodies.
Among the questions remaining to be answered more definitively about the effects of lead
on the kidneys is the lowest blood lead level at which renal effects occur. In this regard it
should be noted that recent studies in humans have indicated that the EDTA lead-mobilization
test is the most reliable technique for detecting persons at risk for chronic nephropathy;
blood lead measurements are a less satisfactory indicator because they may not accurately
reflect cumulative absorption some time after exposure to lead has terminated. Other ques-
tions include the following: Can a distinctive lead-induced renal lesion be identified either
in functional or histologic terms? What biologic measurements are most reliable for the pre-
diction of lead-induced nephropathy? What is the incidence of lead nephropathy in the general
population as well as among specifically defined subgroups with varying exposure? What is the
natural history of treated and untreated lead nephropathy? What is the mechanism of lead-
induced hypertension and renal injury? What are the contributions of environmental and
genetic factors to the appearance of renal injury due to lead? Conversely, the most difficult
question of all may well be to determine the contribution of low levels of lead exposure to
possible exacerbation of renal disease of non-lead etiologies.
12.10.6 Effects of Lead on Reproduction and Development
The most clear-cut data described in this section on reproduction and development are
derived from studies employing high lead doses in laboratory animals. There is still a need
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for more critical research to evaluate the possible subtle toxic effects of lead on the fetus,
using biochemical, ultrastructural, or behavioral endpoints. An exhaustive evaluation of
lead-associated changes in offspring should include consideration of possible effects due to
paternal lead burden as well. Neonatal lead intake via consumption of milk from lead-exposed
mothers may also be a factor at times. Moreover, it must be recognized that lead's effects on
reproduction may be exacerbated by other environmental factors (e.g., dietary influences,
maternal hyperthermia, hypoxia, and co-exposure to other toxins).
There are currently no reliable data pointing to adverse effects in human offspring fol-
lowing lead exposure of fathers per se. Early studies of pregnant women exposed to high
levels of lead indicated toxic, but not teratogenic, effects on the conceptus. Unfortunately,
the collective human data regarding lead's effects on reproduction or rn utero development
currently do not lend themselves to accurate estimation of exposure-effect or no-effect
levels. This is particularly true regarding effects on reproductive performance in women,
which have not been well documented at low exposure levels. Still, prudence would argue
for avoidance of lead exposures resulting in blood lead levels exceeding 25-30 ug/dl -jn
pregnant women or women of child-bearing age in general, given the equilibration between
maternal and fetal blood lead concentrations that occurs and the growing evidence for dele-
terious effects in young children as b'lood lead levels approach or exceed 25-30 ug/dl. In-
dustrial exposure of men to lead at levels resulting in blood lead values of 40-50 ug/dl also
appear to result in altered testicular function.
The paucity of human exposure data forces an examination of the animal studies for indi-
cations of threshold levels for effects of lead on the conceptus. It must be noted that the
animal data are almost entirely derived from rodents. Based on these rodent data, it seems
likely that fetotoxic effects have occurred in animals at chronic exposures to 600-800 ppm
inorganic lead in the diet. Subtle effects appear to have been observed at 5-10 ppm in the
drinking water, while effects of inhaled lead have been seen at levels of 10 mg/m3. With mul-
tiple exposure by gavage, the lowest observed effect level is 64 mg/kg per day, and for expo-
sure via injection, acute doses of 10-16 mg/kg appear effective. Since humans are most likely
to be exposed to lead in their diet, air, or water, the data from other routes of exposure are
of less value in estimating harmful exposures. Indeed, it appears that teratogenic effects
occur in experimental animals only when the maternal dose is given by injection.
Although human and animal responses may be dissimilar, the animal evidence does document
a variety of effects of lead exposure on reproduction and development. Measured or apparent
changes in production of or response to reproductive hormones, toxic effects on the gonads,
and toxic or teratogenic effects on the conceptus have all been reported. The animal data
also suggest subtle effects on such parameters as metabolism and cell structure that should be
monitored in human populations. Well-designed prospective human epidemiological studies
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involving large numbers of subjects are still needed (beyond the few currently available).
Such data could clarify the relationship of exposure periods, exposure durations, and blood
lead concentrations associated with significant effects and are needed for estimation of no-
effect levels as well. (Recent studies, most of which are prospective epidemiological inves-
tigations, on the relationship between relatively low-level lead exposure and effects on fetal
and child development, along with supporting experimental evidence on possible underlying
mechanisms, are reviewed in an Addendum to this document.)
12.10.7 Genotoxic and Carcinogenic Effects of Lead
It is difficult to conclude what role lead may play in the induction of human neoplasia.
Epidemiological studies of lead-exposed workers provide no definitive findings. However, sta-
tistically significant elevations in cancer of the respiratory tract and digestive system in
workers exposed to lead and other agents warrant some concern. Since it is clear that lead
acetate can produce renal tumors in some experimental animals, it seems reasonable to conclude
that at least this particular lead compound should be regarded as a carcinogen and prudent to
treat it as if it were also a human carcinogen (as concluded by the International Agency for
Research on Cancer). However, this statement is qualified by noting that lead has been seen
to increase tumorigenesis rates in animals only at relatively high concentrations, and there-
fore does not seem to be a potent carcinogen. Jin vitro studies further support the genotoxic
and carcinogenic role of lead, but also indicate that lead is not potent in these systems.
12.10.8 Effects of Lead on the Immune System
Lead renders animals more susceptible to endotoxins and infectious agents. Host suscep-
tibility and the humoral immune system appear to be particularly sensitive. As postulated in
recent studies, the macrophage may be the primary immune target cell of lead. Lead-induced
immunosuppression occurs in experimental animals at low lead exposures that, although not in-
ducing overt toxicity, may nevertheless be detrimental to health. Available data provide good
evidence that lead affects immunity, but additional studies are necessary to elucidate the
actual mechanisms by which lead exerts its immunosuppressive action. Knowledge of the effects
of lead on the human immune system is lacking and must be ascertained in order to determine
permissible levels for human exposure. However, in view of the fact that lead affects im-
munity in laboratory animals and is immunosuppressive at very low dosages, its potential for
serious effects in humans should be carefully considered.
12.10.9 Effects of Lead on Other Organ Systems
The cardiovascular, hepatic, gastrointestinal, and endocrine systems generally show signs
of dysfunction mainly at relatively high lead exposure levels. Consequently, in most clinical
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and experimental studies, attention has been primarily focused on more sensitive and vulner-
able target organs, such as the hematopoietic and nervous systems. However, some work does
suggest that humans and animals show significant increases in blood pressure following chronic
exposure to low levels of lead (see Addendum to this document for a detailed discussion of the
relationship between blood lead and blood pressure and the possible biological mechanisms
which may be responsible for this association). It should also be noted that overt gastroin-
testinal symptoms associated with lead intoxication have been observed to occur in lead
workers at blood lead levels as low as 40-60 M9/dl- These findings suggest that effects on
the gastrointestinal and cardiovascular systems may occur at relatively low exposure levels,
but remain to be more conclusively demonstrated by further scientific investigations. Current
evidence indicates that various endocrine processes may be affected by lead at relatively high
exposure levels. Little information exists on endocrine effects at lower exposure levels,
except for alterations in vitamin-D metabolism previously discussed as secondary to heme syn-
thesis effects and occurring at blood lead levels ranging below 30 ug/dl to as low as 12
ug/dl. (Evidence relating endocrine function to various recently reported lead-associated
effects on human fetal and child development, including effects on growth and stature, is
reviewed in the Addendum to this document.)
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12-370
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APPENDIX 12-A
SUMMARY OF PSYCHOMETRIC TESTS USED TO ASSESS COGNITIVE
AND BEHAVIORAL DEVELOPMENT IN PEDIATRIC POPULATIONS
12A-1
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TABLE 12A. TESTS COMMONLY USED IN A PSYCHO-EDUCATIONAL BATTERY FOR CHILDREN
Age range
Noras
Scores
Advantages
Disadvantages
General Intelligence Tests
Stanford-Binet (form L-H)
2 yrs - Adult 1972
Wechsler Preschool & Primary 4 - 6% yrs 1967
Scales of Intelligence (WPPSI) Best for 5-yr-olds
Wechsler Intelligence Scale 6-16 yrs
for Children-Revised (WISC-R)
PO
McCarthy Scales of Children's
Abilities (MSCA)
ft - Wi yrs
Best for ages
4-6
1974
1972
Bayley Scales of Mental
Development
2-30 mos.
1969
1. Deviation IQ:
Mean = 100 SD = 16
2. Mental Age Equivalent
1. Deviation IQ:
Mean = 100 SD = IS
2. Scaled Scores for
10 sub tests:
Mean = 10 SO = 3
1. Deviation IQ:
Mean = 100 SD = IS
2. Scaled Scores for
10 subtests: Mean = 10
SD= 3
1. General Cognitive Index:
Mean = 100 SD = 16
2. Scaled scores for 5
subtests: wan = SO
SD = 10 Age equivalents
can be derived.
1. Standard scores
(H = 100 SO = 16)
2. Mental Development
PsychoMotor Index
1. Good reliability & validity
2. Predicts school performance
3. Covers a wide age range
1. Good reliability & validity
2. Predicts school performance
3. Gives a profile of verbal &
non-verbal skills.
4. Useful in early identifica-
tion of learning disability
1. Good reliability & validity
2. Predicts school performance
3. Gives a profile of verbal
and non-verbal skills
4. Useful in identification of
learning disability
1. Good reliability & validity
2. Good predictor of school
performance
3. Useful in identification of
learning disabilities when
given with a WISC-R or
Stanford-Binet
4. Gives good information for
educational programming
1. Norms are excellent
2. Satisfactory reliability
and validity
3. Best measure of infant
development
1. Tests mostly verbal skills
especially after 6 yrs
2. Does not give a profile
of skills
1. Narrow age range
2. Mentally retarded children
find this a disproportionately
difficult test
1. Gives a lower IQ than
Stanford-Binet for normal
and bright children
1. Children score much lower
than on WISC-R or
Stanford-Binet
2. Narrow age range
1. Not a good predictor of
later functioning in
average as in below average
children
-------
TABLE 12A. (continued)
Age range
Norms
Scores
Advantages
Disadvantages
Slosson Intelligence Test
Infancy - 27 yrs 1963
1. Ratio IQ: is not
related to general
population
Peabody Picture Vocabulary
Test
2>s - 18 yrs
1959,rev.1981 1. Verbal IQ
White, 2. Age equivalent
Middle class
sample
(__ Visual-Hotor Tests
r\j
:> Frostig Developmental Test of
-------
TABLE 12A. (continued)
Age range
Horns
Scores
Advantages
Disadvantages
Educational Tests
Wide Range Achievement Test
(WRAT)
Peabody Individual
Achievement Test (PIAT)
5 yrs - Adult
5-18 yrs
1976
Revised
1969
-fc.
Woodcock Reading
Mastery Tests
Spache Diagnostic
Reading Scales
Key Hath Diagnostic
Arithmetic Test
Kgn - 12 grade
1st - 8th grade
1971-72
adjusted for
social class
1972
Pre-school - 6th
grade
1971
1. Standard Score:
•ean = 100 SO = 15
Z. Grade equivalent
1. Standard Scores:
Mean = 100 SD = 15
2. Grade equivalent
3. Age equivalent
1. Grade equivalent
2. Standard Score
3. Percent!le Rank
1. Instructional level of
reading (grade equiva-
lent).
2. Independent level of
reading.
3. Potential level of
reading
1. Grade equivalent
1. Good reliability & validity
Reading scores predict
grade level
2. Tasks similar to actual
work
1. Reading portion tests
word recognition only
2. Responses require good
organizational skills
(could be an advantage)
1. Tests word recognition and 1.
2. Breaks down skills into 5
areas 2.
1. Good reliability
2. Breakdown of reading skills
useful diagnostically and in
planning remediation
3. Easy to administer and score
2.
Independent level score
predicts gains following
remediation
Good breakdown of reading
skills
Excellent breakdown of math
skill*
Easy to administer and
score
Moderate reliability. Low
stability for Kindergarten
No data on predictive
validity
A multiple choice test
requiring child to recog-
nize correct answer (could
be an advantage).
Heavily loaded on verbal
reasoning.
Factor structure changes
with age.
No data on validity
Fairly complex scoring
Moderate reliability
3. No good data on validity
1. Moderate reliability
2. No data on validity
-------
TABLE 12A. (continued)
Age range
Nor
Scores
Advantages
Disadvantages
Tests of Adaptive functioning
Vineland Social Maturity Scale Birth - 25 yrs
AAHD Adaptive Behavior Scale 3 yrs - Adult
1983 1. Social Quotient (Ratio)
Revised 2. Social Age Equivalent
1974
Institu-
tionalized
Retardates;
Public School
Children (1982)
1. Percent!le Ranks
2. Scaled scores
Progress Assessment Chart of
Social Development (PAC)
Developmental Profile
Conners Rating Scale
Birth - Adult
Birth - 12 yrs
3 yrs - 17 yrs
1976
1972
1978
No Scores
1. Age equivalents in 5
5 areas
2. IQ equivalency (IQE)
1. Age equivalents
1. Easily administered
2. Good reliability for normal
and MR chidren
1. Diserial nates between EMR
and regular classes
2. Useful for class placement
and Monitoring progress
1. Useful for training and
assessing progress
2. Gives profile of skills
1. Good reliability and valid-
ity. Excellent study of
construct validity reported
in Manual.
2. Gives a profile of skills
1. Most widely used Measure of
attention deficit disorder
2. Four factors: conduct prob-
lems; hyperactivity;
inattentive-passive; hyper-
activity index
1. Poor norms
2. No data on validity
3. Items are limited past
preschool years
4. Scores decrease with age
for MR children
1. Moderate reliability for
independent living skills
scale. Poor reliability
for ma1adaptive behaviour
scale.
2. Lengthy administration
3. Items & scoring are not
behaviorally objective
1. No data on reliability or
validity
1. IQE underestimates IQ of
above average children,
overestimates IQ of below
average chiIdren.
Parents' ratings don't pre-
dict as well as teachers'
ratings
Works best Middle class
children
Werry-Weiss-Peters Hyperactivity 1 yr - 9 yrs
Scale
1974, 1977 1. Age equivalents
1. Good measure of hyperac-
tivity
2. Seven Factors
1. Limited age range
2. Standardized on Middle
class children
-------
13. EVALUATION OF HUMAN HEALTH RISKS ASSOCIATED WITH
EXPOSURE TO LEAD AND ITS COMPOUNDS
13.1 INTRODUCTION
This chapter attempts to integrate, concisely, key information and conclusions discussed
in preceding chapters into a coherent framework by which interpretation and judgments can be
made concerning the risk to human health posed by present levels of lead contamination in the
United States.
Towards this end, the chapter is organized into eight sections, each of which discusses
one or more of the following major components of the overall health risk evaluation: (1) ex-
ternal and internal exposure aspects of lead; (2) lead metabolism, which determines the rela-
tionship of external lead exposure to associated health effects of lead; (3) qualitative and
quantitative characterization of key health effects of lead; and (4) identification of popula-
tion groups at special risk for health effects associated with lead exposure.
The various aspects of lead exposure discussed include: (1) an historical perspective on
the input of lead into the environment as well as the nature and magnitude of current lead
input; (2) the cycling of lead through the various environmental compartments; and (3) levels
of lead in those media most relevant to lead exposure of various segments of the U.S. popula-
tion. These various aspects of lead exposure are summarized in Section 13.2.
In regard to lead metabolism, some of the relevant issues addressed include: (1) the
major quantitative characteristics of lead absorption, distribution, retention, and excretion
in humans and how these differ between adults and children; (2) the toxicokinetic bases for
external/internal lead exposure relationships with respect to both internal indicators and
target tissue lead burdens; and (3) the relationships between internal and external indices of
lead exposure, i.e., blood lead levels, and lead concentrations in air, food, water, and dust/
soil. Section 13.3 summarizes the most salient features of lead metabolism, whereas Section
13.4 addresses experimental and epidemiological data concerning various blood lead-environ-
mental media lead relationships.
In discussion of the various health effects of lead, the main emphasis is on the identi-
fication of those effects most relevant to various segments of the general U.S. population and
the placement of such effects in a dose-effect/dose-response framework. With regard to the
latter, a crucial issue has to do with relative response of various segments of the population
in terms of observed effect levels as indexed by some exposure indicator. Furthermore, it is
of interest to assess the extent to which available information supports the existence of a
continuum of effects as one proceeds across the spectrum of exposure levels. Discussion of
13-1
-------
data on the relative number or percentage of members (i.e., "responders") of specific popula-
tion groups that can be expected to experience a particular effect at various lead exposure
levels is also important in order to permit delineation of dose-response curves for the
relevant effects in different segments of the population. These matters are discussed in
Sections 13.5 and 13.6.
Melding of information from the sections on lead exposure, metabolism, and biological
effects permits the identification of population segments at special risk in terms of physio-
logical and other host characteristics, as well as heightened vulnerability to a given effect;
these risk groups are discussed in Section 13.7. With demographic identification of indivi-
duals at risk, one may then draw upon population data from other sources to obtain a numerical
picture of the magnitude of population groups at potential risk. This is also discussed in
Section 13.7.
Section 13.8 summarizes key information and conclusions derived from the analyses
presented in the preceeding sections.
13.2 EXPOSURE ASPECTS
13.2.1 Sources of Lead Emission in the United States
The most important issues addressed here concerning the sources of lead in the human
environment are: What additional pathways of human consumption have been added in the course
of civilization? What are the relative contributions of natural and anthropogenic lead? From
the available data, what trends can be expected in the potential consumption of lead by
humans? What is the impact of normal lead cycling processes on total human exposure? And,
finally, are there population segments particularly at risk due to a higher-potential expo-
sure?
Figure 13-1 is a composite of similar figures appearing in Chapters 7 and 11. This
figure shows that four of the five sources of lead in the human environment are of anthropo-
genic origin. The only significant natural source is from the geochemical weathering of
parent rock material as an input to soils. Of the four anthropogenic pathways, two are close-
ly associated with atmospheric emissions and two (pigments and solder) are more directly
related to the use of metallurgical compounds in products consumed by humans.
It is clear that natural sources contribute only a very small fraction to total lead in
the biosphere. Levels of lead in the atmosphere, the main conduit for lead movement from
sources into various environmental compartments, are 10,000 to 20,000-fold higher in some
urban areas than in the most remote regions of the earth. Chronological records assembled
13-2
-------
INDUSTRIAL
EMISSIONS
CRUSTAL
WEATHERING
SURFACE AND
GROUND WATER
FECES URINE
Figure 13-1. Pathways of laad from the environment to man, main
compartments involved in partitioning of internal body burden of
absorbed/retained lead, and main routes of leed excretion.
13-3
-------
using reliable lead analysis techniques show that atmospheric lead levels were at least 2,000-
fold lower than at present before abrupt anthropological inputs accelerated with the indus-
trial revolution and, more recently, with the introduction of leaded gasoline. For actual
comparison, estimates indicate a general background air lead level of 0.00005-0.0005 ug Pb/m3
versus current urban air lead concentrations frequently approaching 1.0 ug Pb/m3. A recent
measurement of 0.000076 ug Pb/m3 at the South Pole, using highly reliable lead analyses, sug-
gests an anthropogenic enrichment factor of 13,000-fold compared to the same urban air level
of 1.0 ug Pb/m3.
Lead occupies an important niche in the U.S. economy, with consumption averaging 1.28 x
106 metric tons/year over the period 1971-1984. Of the various categories of lead consump-
tion, those of pigments, gasoline additives, ammunition, foil, solder, and steel products are
widely dispersed and therefore unrecoverable. In the United States, about 39,000 tons are
emitted to the atmosphere each year, including 35,000 tons as gasoline additives. Lead and
its compounds enter the atmosphere at various points during mining, smelting, processing, use,
recycling, or disposal. Leaded gasoline combustion in vehicles accounted for 90 percent of
the total anthropogenic input into the atmosphere in the United States in 1984; of the remain-
ing 10 percent of total emissions from stationary sources, 5 percent was from the metallurgical
industry, 3 percent was from waste combustion, 1 percent from combustion during energy produc-
tion, and 1 percent was from miscellaneous sources. Atmospheric emissions have declined in
recent years with the phase-down of lead in gasoline.
The fate of emitted particulate lead depends on particle size. It has been estimated
that, of the 75 percent of combusted gasoline lead which immediately departs the vehicle in
exhaust, 46 percent is in the form of particles <0.25 urn mass median aerodynamic diameter
(MMAD) and 54 percent has an average particle size >10 urn. The sub-micron fraction is in-
volved in long-range transport, whereas the larger particles settle mainly near the roadway.
13.2.2 Environmental Cycling of Lead
The atmosphere is the main conduit for movement of lead from emission sources to other
environmental compartments. Removal of lead from the atmosphere occurs by both wet and dry
deposition processes, each mechanism accounting for about one-half of the atmospheric lead
removed. The fraction of lead emitted as alkyl lead vapor (1-6 percent) undergoes subsequent
transformation to other, more stable compounds such as triethyl- or trimethyl lead as a com-
plex function of sunlight, temperature, and ozone level.
Studies of the movement of lead emitted into the atmosphere indicate that air lead levels
decrease logarithmically with distance away from the source: (1) along gradients from emis-
sion sites, e.g., roadways and smelters; (2) within urban regions away from central business
districts; (3) from urban to rural areas; and (4) from developed to remote areas.
13-4
-------
By means of wet and dry deposition, atmospheric lead is transferred to terrestrial sur-
faces and bodies of water. Transfer to water occurs either directly from the atmosphere or
through runoff from soil to surface waters. A sizeable fraction of water-borne lead becomes
lodged in aquatic sediments. Percolation of water through soil does not transport much lead
to ground water because most of the lead is retained at the soil surface.
The fate of lead particles on terrestrial surfaces depends upon such factors as the
mechanism of deposition, the chemical form of the particulate lead, the chemical nature of the
receiving soil, and the amount of vegetation cover. Lead deposited on soils is apparently
immobilized by binding to humic or fulvic acids, or by ion exchange on clays and hydrous
oxides. In industrial, playground, and household environments, atmospheric particles accumu-
late as dusts with lead concentrations often greater than 1000 ug/g. It is important to dis-
tinguish these dusts from windblown soil dust, which typically has a lead concentration of
10-30 ug/g.
It has been estimated that soils adjacent to roadways have been enriched in lead content
by as much as 10,000 ug/g soil since 1930, while in urban areas and sites adjacent to smelters
as much as 130,000 ug/g has been measured in the upper 2-5 cm layer of soil.
Soil appears to be the major sink for emitted lead, with a residency half-time of de-
cades; however, soil as a reservoir for lead cannot be considered as an infinite sink, because
lead will continue to pass into the grazing and detrital food chains and sustain elevated lead
levels in them until equilibrium is reached. It was estimated in Chapters 7 and 8 that soils
not adjacent to major sources such as highways and smelters contain 3-5 ug/g of anthropogenic
lead, and that this lead has caused an increase of lead in soil moisture by a factor of 2-4.
Thus, movement of lead from soils to other environmental compartments is at least twice the
prehistoric rate and will continue to increase for the foreseeable future.
Lead enters the aquatic compartment by direct transfer from the atmosphere via wet and
dry deposition, as well as indirectly from the terrestrial compartment via surface runoff.
Water-borne lead, in turn, may be retained in some soluble fraction or may undergo sedimenta-
tion, depending on such factors as pH, temperature, suspended matter which may entrap lead,
etc. Present levels of lead in natural waters represent a 50-fold enrichment over background
content, from 0.02 to 1.0 pg/1, due to anthropogenic activity. Surface waters receiving urban
effluent represent a 2500-fold and higher enrichment (50 ug Pb/1 and higher).
13.2.3 Levels of Lead in Various Media of Relevance to Human Exposure
Human populations in the United States are exposed to lead in air, food, water, and dust.
In rural areas, Americans not occupationally exposed to lead are estimated to consume 40-60 ug
Pb/day. This level of exposure is referred to as the baseline exposure for the American
population because it is unavoidable except by drastic change in lifestyle or by regulation of
13-5
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lead in foods or ambient air. There are several environmental circumstances that can increase
human exposures above baseline levels. Most of these circumstances involve the accumulation
of atmospheric dusts in the work and play environments. A few, such as pica and family home
gardening, may involve consumption of lead in chips of exterior or interior house paint.
13.2.3.1 Ambient Air Lead Levels. Monitored ambient air lead concentration values in the
United States are contained in two principal data bases: (1) EPA's National Air Sampling Net-
work (NASN), recently renamed National Filter Analysis Network (NFAN); and (2) EPA's National
Aerometric Data Bank, consisting of measurements by state and local agencies in conjunction
with compliance monitoring for the current ambient air lead standard.
NASN data for 1982, the latest year in the annual surveys for which valid distinctions
can be made between urban and non-urban stations, indicate that most urban sites show reported
annual averages below 0.7 ug Pb/m3, while the majority of non-urban locations have annual
figures below-0.2 ug Pb/m3. Over the interval 1976-1984, there has been a downward trend in
these averages, mainly attributable to decreasing lead content of leaded gasoline and the
increasing usage of lead-free gasoline. Furthermore, examination of quarterly averages over
this interval shows a typical seasonal variation, characterized by maximum air lead values in
summer and minimum values in winter.
With respect to the particle size distribution of ambient air lead, EPA studies using
cascade impactors in six U.S. cities have indicated that 60-75 percent of such air lead was
associated with sub-micron particles. This size distribution is significant in considering
the distance particles may be transported and the deposition of particles in the pulmonary
compartment of the respiratory tract. The relationship between airborne lead at the monitor-
ing station and the lead inhaled by humans is complicated by such variables as vertical gradi-
ents, relative positions of the source, the monitor, and the person, and the ratio of indoor
to outdoor lead concentrations. Personal monitors would probably be the most effective means
to obtain an accurate picture of the amount of lead inhaled during the normal activities of an
individual. However, the information gained would be insignificant, considering that inhaled
lead is generally only a small fraction of the total lead exposure, compared to the lead in
food, beverages, and dust. The critical question in regard to airborne lead is how much lead
becomes entrained in dust. In this respect, the existing monitoring network may provide an
adequate estimate of the air concentration from which the rate of deposition can be deter-
mined.
13.2.3.2 Levels of Lead In Dust. The lead content of dusts can figure prominently in the
total lead exposure picture for young children. Lead in aerosol particles deposited on rigid
surfaces in urban areas (such as sidewalks, porches, steps, parking lots, etc.) does not
undergo dilution compared to lead transferred by deposition onto soils. Lead in dust can
13-6
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approach extremely high concentrations and can accumulate in the interiors of dwellings as
well as in the outside surroundings, particularly in urban areas.
Measurements of soil lead to a depth of 5 cm in areas of the United States were shown in
one study to range from 150 to 500 ug/g dry weight close to roadways (i.e., within 8 meters).
By contrast, lead in dusts deposited on or near heavily traveled traffic arteries show levels
in major U.S. cities ranging up to 8000 ug/g and higher. In residential areas, exterior dust
lead levels are approximately 1000 ug/g or less if contaminated only by atmospheric lead.
Levels of lead in house dust can be significantly elevated; a study of house dust samples in
Boston and New York City revealed levels of 1000-2000 ug/g. Some soils adjacent to houses
with exterior lead-based paints may have lead concentrations greater than 10,000 ug/g.
Forty-four percent of the baseline consumption of lead by children is estimated to result
from consumption of 0.1 g of dust per day (Tables 13-1 and 13-2). Ninety percent of this dust
lead is of atmospheric origin. Dust also accounts for more than 90 percent of the additive
lead attributable to living in an urban environment or near a smelter (Table 13-3).
13.2.3.3 Levels of Lead in Food. The route by which adults and older children in the base-
line population of the United States receive the largest proportion of lead intake is through
foods, with reported estimates of the dietary lead intake for Americans ranging from 35 to 55
ug/day. The added exposure from living in an urban environment is about 28 HQ/day for adults
and 91 ug/day for children, all of which can be attributed to atmospheric lead.
Atmospheric lead may be added to food crops in the field or pasture, during transporta-
tion to the market, during processing, and during kitchen preparation. Metallic lead, mainly
solder, may be added during processing and packaging. Other sources of lead, as yet undeter-
mined, increase the lead content of food between the field and dinner table. American chil-
dren, adult females, and adult males consume 21, 32, and 45 ug Pb/day, respectively, in food
and beverages. Of these amounts, 45-65 percent is of direct atmospheric origin, 25-37 per-
cent is of metallic origin, and 5-8 percent is of undetermined origin.
Processing of foods, particularly canning, can significantly add to their background lead
content, although it appears that the impact of this is being lessened with the trend away
from use of lead-soldered cans. The canning process can increase lead levels 8-to 10-fold
higher than for the corresponding uncanned food items. Home food preparation can also be a
source of additional lead in cases where food preparation surfaces are exposed to moderate
amounts of high-lead household dust.
13.2.3.4 Lead Levels in Drinking Water. Lead in drinking water may result from contamination
of the water source or from the use of lead materials in the water distribution system. Lead
entry into drinking water from the latter is increased in water supplies which are plumbo-
solvent, i.e., with a pH below 6.5. Exposure of individuals occurs through direct ingestion
of the water or via food preparation in such water.
13-7
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TABLE 13-1. SUMMARY OF BASELINE HUMAN EXPOSURES TO LEAD
(ug/day)
I
00
Source
Child-2 yr old
Inhaled air
Food, Water &
beverages
Dust
Total
Percent
Adult female
Inhaled air
Food, Water &
beverages
Oust
Total
Percent
Adult male
Inhaled air
Food, Water &
beverages
Dust
Total
Percent
Total
lead
consumed
0.5
25.1
21.0
46.6
100%
1.0
32.0
4.5
37.5
100%
1,0
45.2
4.5
50.7
100%
Soil
Natural
lead
consumed
0.001
0.71
0.6
1.3
2.8%
0.002
0.91
0.2
1.2
3.1%
0.002
1.42
0.2
1.6
3.1%
Indirect
atmospheric
lead*
-
1.7
-
1.7
3.5%
-
2.4
_
2.5
6.6%
-
3.5
-
3.5
6.8%
Direct
atmospheric
lead*
0.5
10.3
19.0
29.8
64.0%
1.0
12.6
2.9
17.4
46.5%
1.0
19.3
2.9
23.2
45.8%
Lead from
solder or
other metals
-
11.2
-
11.2
24.0%
-
8.2
-
13.5
36.1%
-
18.9
-
18.9
37.2%
Lead of
undetermined
origin
-
1.2
1.4
2.6
5.6%
-
1.5
.1.4
2.9
7.8%
-
2.2
1.4
3.6
7.0%
*Indirect atmospheric lead has been previously incorporated into soil, and will probably remain in the
soil for decades or longer. Direct atmospheric lead has been deposited on the surfaces of vegetation
and living areas or incorporated during food processing prior to human consumption.
Source: This report.
-------
TABLE 13-2. RELATIVE BASELINE HUMAN LEAD EXPOSURES EXPRESSED PER KILOGRAM BODY WEIGHT*
Child (2-yr-old)
Inhaled air
Food and beverages
Dust
Total
Adult female
Inhaled air
Food and beverages
Dust
Total
Adult male
Inhaled air
Food and beverages
Dust
Total
Total
lead
consumed,
ug/day
0.5
25.1
21.0
46.6
1.0
32.0
4.5
37.5
1.0
45.2
4.5
50.7
Total lead consumed
per kg body wt,
ug/kg'day
0.05
2.5
2.1
4.65
0.02
0.64
0.09
0.75
0.014
0.65
0.064
0.73
Atmospheric lead
per kg body wt,
ug/kg-day
0.05
1.0
1.9
2.95
0.02
0.25
0.06
0.33
0.014
0.28
OJD4_
0.334
*Body weights: 2-year-old child = 10 kg; adult female = 50 kg; adult male = 70 kg.
Source: This report.
The major source of lead contamination of drinking water is the distribution system it-
self, particularly in older urban areas. Highest levels are encountered in "first-draw" sam-
ples, i.e., water sitting in the piping system for an extended period of time. In a large
community water supply survey of 969 systems carried out in 1969-1970, it was found that the
prevalence of samples exceeding 0.05 ug/g was greater where water was plumbo-solvent.
Most drinking water, and the beverages produced from drinking water, contain 0.007-
0.011 ug Pb/g. The exceptions are canned juices and soda pop, which range from 0.018 to
0.040 ug/g. About 15 percent of the lead consumed in drinking water and beverages is of
direct atmospheric origin; 60 percent comes from solder and other metals.
13-9
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TABLE 13-3. SUMMARY OF POTENTIAL ADDITIVE EXPOSURES TO LEAD (pg/day)
Baseline exposure:
Child
Inhaled air
Food, water & beverages
Dust
Total baseline
Additional exposure due to:
Urban atmospheres1
Family gardens2
Interior lead paint3
Residence near smelter4
Secondary occupational5
Baseline exposure:
Adult male
Inhaled air
Food, water & beverages
Dust
Total baseline
Additional exposure due to:
Urban atmospheres1
Family gardens2
Interior lead paint3
Residence near smelter4
Occupational6
Secondary occupational5
Smoking7
Wine consumption8
Total
lead
consumed,
pg/day
0.5
25.1
21.0
46.6
91
48
110
880
150
1.0
54.7
4.5
60.2
28
120
17
100
1100
44
30
100
Atmospheric
lead
consumed,
ug/day
0.5
10.3
19.0
29.8
91
12
880
1.0
20.3
2.9
24.2
28
30
100
1100
27
7
Other
lead
sources,
pg/day
-
14.8
2.0
16.8
36
110
-
34.4
1.6
36.0
17
3
7
Includes lead from household (1000 pg/g) and street dust (1500 pg/g) and inhaled air
(0.75 pg/m3).
2Assumes soil lead concentration of 2000 pg/g; all fresh leafy and root vegetables, sweet
corn of Table 7-12 replaced by produce from garden. Also assumes 25% of soil lead is of
atmospheric origin.
3Assumes household dust rises from 300 to 2000 pg/g. Dust consumption remains the same
as baseline.
4Assumes household and street dust increases to 10,000 pg/g.
5Assumes household dust increases to 2400 pg/g.
6Assumes 8-hr shift at 10 pg Pb/m3 or 90% efficiency of respirators at 100 pg Pb/m3, and
occupational dusts at 100,000 pg/m3.
70ne-and-a-half packs per day.
8Assumes unusually high consumption of one liter per day.
Source: This report.
13-10
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13.2.3.5 Lead in Other Media. Flaking lead paint as well as paint chips and weathered pow-
dered paint in and around deteriorated housing stock in urban areas of the Northeast and Mid-
west has long been recognized as a major source of lead exposure for young children residing
1n this housing stock, particularly for children with pica. Census data, for example, indi-
cate that there are approximately 27 million residential units in the United States built
before 1940, many of which still contain lead-based paint. Also, individuals who are ciga-
rette smokers may inhale significant amounts of lead in tobacco smoke. One study has indi-
cated that the smoking of 30 cigarettes daily results in lead intake equivalent to that of
inhaling lead in ambient air at a level of 1.0 ug/m3.
13.2.3.6 Cumulative Human Lead Intake From Various Sources. Table 13-1 shows the baseline of
human lead exposures in the United States as described in detail in Chapter 7. These data
show that atmospheric lead accounts for at least 45 percent of the baseline adult consumption
and 60 percent of the daily consumption by a 2-yr-old child. These percentages are conserva-
tive estimates because a part of the lead of undetermined origin may originate from atmos-
pheric lead not yet accounted for.
From Table 13-2, it can be seen that young children have a dietary lead intake rate that
is 5-fold greater than for adults, on a body weight basis. To these observations must be
added that absorption rates for lead are higher in children than in adults by at least 3-fold.
Overall, then, the rate of lead entry into the blood stream of children, on a body weight
basis, is estimated to be twice that of adults from the respiratory tract and six to nine
times greater from the GI tract. Since children consume more dust than adults, the atmos-
pheric fraction of the baseline exposure is sixfold higher for children than for adults, on a
body weight basis. These differences generally tend to place young children at greater risk,
in terms of relative amounts of atmospheric lead absorbed per kg body weight, than adults
under any given lead exposure situation.
13.3 LEAD METABOLISM: KEY ISSUES FOR HUMAN HEALTH RISK EVALUATION
From the detailed discussion of those various quantifiable characteristics of lead toxi-
cokinetics in humans and animals presented in Chapter 10, several clear issues emerge as being
important for full evaluation of the human health risk posed by lead:
(1) Differences in systemic or internal lead exposure of groups within the general popu-
lation in terms of such factors as age/development and nutritional status; and
(2) The relationship of indices of internal lead exposures to both environmental levels
of lead and tissues levels/effects.
13-11
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Item 1 is used along with additional information on relative sensitivity to lead health
effects to provide the basis for identifying segments within human populations at increased
risk in terms of exposure criteria. Item 2 deals with the adequacy of current means for
assessing internal lead exposure in terms of providing adequate margins of protection from
lead exposures which produce health effects of concern.
13.3.1 Differential Internal Lead Exposure Within Population Groups
Compared to adults, young children take in more lead through the gastrointestinal and
respiratory tracts on a unit body weight basis, absorb a greater fraction of this lead intake,
and also retain a greater proportion of the absorbed amount. Unfortunately, such amplifica-
tion of these basic toxicokinetic parameters in children versus adults also occurs at the time
when: (1) humans are developmentally more vulnerable to the effects of toxicants such as lead
in terms of metabolic activity; and (2) the interactive relationships of lead with such fac-
tors as nutritive elements are such as to induce a negative course toward further exposure
risk.
Typical of physiological differences in children versus adults in terms of lead exposure
implications is a more metabolically active skeletal system in children. In children, turn-
over rates of bone elements such as calcium and phosphorus are greater than in adults, with
correspondingly greater mobility of bone-sequestered lead. This activity is a factor in the
observation that the skeletal system of children is relatively less effective as a depository
for lead than in adults.
Metabolic demand for nutrients, particularly calcium, iron, phosphorus, and the trace
elements is such that widespread deficiencies of these nutrients exist, particularly among
poor children. The interactive relationships of all of these elements with lead are such that
deficiency states enhance lead absorption and/or retention. In the case of lead-induced
reductions in 1,25-dihydroxyvitamin D, furthermore, there may exist an increasingly adverse
interactive cycle between lead effects on 1,25-dihydroxyvitamin D and associated increased
absorption of lead.
Quite apart from the physiological differences which enhance internal lead exposure in
children is the unique relationship of 2- to 3-year-olds to their exposure setting by way of
normal mouthing behavior and the extreme manifestation of this behavior, pica. This behavior
occurs in the same age group which studies have consistently identified as having a peak in
blood lead levels. A number of investigations have addressed the quantification of this
particular route of lead exposure, and it is by now clear that such exposure will dominate
other routes when the child's surroundings, e.g., dust and soil, are significantly contami-
nated by lead.
13-12
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Information provided in Chapter 10 also makes it clear that lead traverses the human pla-
cental barrier, with lead uptake by the fetus occurring throughout gestation. Such uptake of
lead poses a potential threat to the fetus via an impact on the embryological development of
the central nervous and other systems. Hence, the only logical means of protecting the fetus
from lead exposure is exposure control during pregnancy. Within the general population, then,
young children and pregnant women qualify as well-defined groups at high risk for lead expo-
sure.
In addition, certain emerging information (noted in Section 13.5 and described in detail
in the Addendum to this document) indicates that increases in blood pressure are associated
with blood lead concentrations ranging from >30-40 ug/dl down to possibly as low as 7 ug/dl;
this association appears to be particularly robust in white males, aged 40-59. Occupational
exposure to lead, particularly among lead workers, logically defines these individuals as also
being in a high-risk category; work place contact is augmented by those same routes and levels
of lead exposure affecting the rest of the adult population. From a biological point of view,
lead workers do not differ from the general adult population with respect to the various
toxicokinetic parameters and any differences in exposure control—occupational versus non-
occupational populations—as they exist are based on factors other than toxicokinetics.
13.3.2 Indices of Internal Lead Exposure and Their Relationship To External Lead Levels and
Tissue Burdens/Effects
Several joints are of importance to consider in the area of lead toxicokinetics: (1) the
temporal characteristics of indices of lead exposure; (2) the relationship of these indicators
to external lead levels; (3) the validity of indicators of exposure in reflecting target tis-
sue burdens; (4) the interplay between these indicators and lead in body compartments; and (5)
those various aspects of this issue that, in particular, refer to children.
At this time, blood lead is widely held to be the most convenient, if imperfect, index of
both lead exposure and relative risk for various adverse health effects. In terms of expo-
sure, however, it is generally accepted that blood lead is a temporally variable measure which
yields an index of relatively recent exposure because of the rather rapid clearance of absor-
bed lead from the blood. Such a measure, then, is of limited usefulness in cases where expo-
sure is variable or intermittent over time, as is often the case with pediatric lead exposure.
Mineralizing tissue (specifically deciduous teeth), on the other hand, accumulate lead over
time in proportion to the degree of lead exposure, and analysis of this material provides an
assessment integrated over a greater time period.
These two methods of assessing internal lead exposure have obvious shortcomings. A blood
lead value will say little about any excessive lead intake at early periods, even though such
remote exposure may have resulted in significant injury. On the other hand, whole tooth or
13-13
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dentine analysis is retrospective in nature and can only be done after the particularly vulne-
rable age in children—under 4-5 years—has passed. Such a measure, then, provides little
utility upon which to implement regulatory policy or clinical intervention.
It may be possible to resolve the dilemmas posed by these existing methods by iji situ
analysis of teeth and bone lead, such that the intrinsic advantage of mineral tissue as a
cumulative index is combined with measurement which is temporally concordant with on-going
exposure. Work in several laboratories offers promise for such iji situ analysis (See Chapters
9 and 10).
A second issue concerning internal indices of exposure to environmental lead is the rela-
tionship of changes in lead content of some medium with changes in blood content. Much of
Chapter 11 was given over to description of the mathematical relationships of blood lead with
lead in some external medium—air, food, water, etc.--without consideration of the biological
underpinnings for these relationships.
Over a relatively broad range of lead exposure through some medium, the relationship of
lead in the external medium to lead in blood is curvilinear, such that relative change in
blood lead per unit change in medium level generally becomes increasingly less as exposure in-
creases. This behavior may reflect changes in tissue lead kinetics, reduced lead absorption,
or increased excretion. With respect to changes in body lead distribution, the relative
amount of whole blood lead in plasma increases significantly with increasing whole blood lead
content; i.e., the plasma/erythrocyte ratio increases. Limited animal data would suggest that
changes in absorption may be one factor in this phenomenon. In any event, modest changes in
blood lead levels with exposure at the higher end of this range are in no way to be taken as
reflecting concomitantly modest changes in body or tissue lead uptake. Evidence continues to
accumulate which suggests that an indicator such as blood lead is an imperfect measure of
tissue lead burdens and of changes in such tissue levels in relation to changes in external
exposure (see Figure 13-2).
In Chapter 10, it was pointed out that blood lead is logarithmically related to chelata-
ble lead (the latter being a more useful measure of the potentially toxic fraction of body
lead), such that a unit change in blood lead is associated with an increasingly larger amount
of chelatable lead. One consequence of this relationship is that moderately elevated blood
lead values will tend to mask the "margin of safety" in terms of mobile body lead burdens.
Such masking is apparent in several studies where chelatable lead levels in children showing
moderate elevations in blood lead overlapped those obtained in subjects showing frank plum-
bism, i.e., overt lead intoxication. In a multi-institutional survey involving several hun-
dred children, it was found that a significant percentage of children with moderately elevated
blood lead values had chelatable lead burdens which qualified them for medical treatment.
13-14
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lout
Figure 13-2. Illustration of main body compartments involved in partitioning, retention, and excretion
of absorbed lead and selected target organs for lead toxicity. Inhaled and ingested lead circulates via
blood (1) to mineralizing tissues such as teeth and bone (2), where long-term retention occurs reflective of
cumulative past exposures. Concentrations of lead in blood circulating to "soft tissue" target organs
such as brain (3), peripheral nerve, and kidney, reflect both recent external exposures and lead re-
circulated from internal reservoirs (e.g. bone). Blood lead levels used to index internal body lead
burden tend to be in equilibrium with lead concentrations in soft tissues and, below 30 /jg/dl, also
generally appear to reflect accumulated lead stores. However, somewhat more elevated current blood
lead levels may "mask" potentially more toxic elevations of retained lead due to relatively rapid declines
in blood lead in response to decreased external exposure. Thus, provocative chelation of some children
with blood leads of 30-40 ^g/dl. for example, results in mobilization of lead from bone and other
tissues into blood and movement of the lead (4) into kidney (5), where it is filtered into urine and
excreted (6) at concentrations more typical of overtly lead-intoxicated children with higher blood lead
concentrations,
13-15
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Related to the above is the question of the source of chelatable lead. It was noted in
Chapter 10 that some sizable fraction of chelatable lead is derived from bone and that this
compels reappraisal of the notion that bone is an "inert sink" for otherwise toxic body lead.
The notion of bone lead as lexicologically inert never did accord with what was known from
studies of bone physiology, i.e., that bone is a "living" organ. The thrust of recent studies
of chelatable lead, as well as interrelationships of lead and bone metabolism, supports the
view that bone lead is actually an insidious source of long-term systemic lead exposure rather
than a protective mechanism which permits significant lead contact in industrialized popula-
tions.
The complex interrelationships of lead exposure, blood lead, and lead in body compart-
ments is of particular interest in considering the disposition of lead in young children.
Since children take in more lead on a weight basis, and absorb and retain more of this lead
than the adult, one might expect that either tissue and blood levels would be significantly
elevated or that the child's skeletal system would be more efficient in lead sequestration.
Average blood lead levels in young children are generally either similar to adult males or
somewhat higher than for adult females. Limited autopsy data, furthermore, indicate that soft
tissue levels in children are not markedly different from adults, whereas the skeletal system
shows an approximate 2-fold increase in lead concentration from infancy to adolescence.
Neglected in this observation is the fact that the skeletal system in children grows at an
exponential rate, so that skeletal mass increases 40-fold during the interval in childhood
when bone lead levels increase 2-fold; this results in an actual increase of approximately 80-
fold in total skeletal lead. If the skeletal growth factor is taken into account, along with
growth in soft tissue and the expansion of vascular fluid volumes, the question of lead dis-
position in children is better understood. Finally, limited animal data indicate that blood
lead alterations with changes in lead exposure are poor indicators of such changes in target
tissue. Specifically, it appears that abrupt reduction of lead exposure will be more rapidly
reflected by decreases in blood lead than by decreased lead concentrations in such target
tissues as the central nervous system, especially in the developing organism. This discord-
ance may underlie the observation that severe lead neurotoxicity in children is associated
with a rather broad range of blood lead values (see Section 12.4).
The above discussion of some of the problems with the use of blood lead in assessing tar-
get tissue burdens or the lexicologically active fraction of total body lead is really a sum-
mary of the toxicokinetic problems inherent with use of blood lead levels in defining margins
of safety for avoiding internal exposure or undue risk of adverse effects. If, for example,
blood lead levels of 30-50 pg/dl in "asymptomatic" children are associated with chelatable
lead burdens which overlap those encountered in frank pediatric plumbism, as documented in
several studies of lead-exposed children, then there is no margin of safety at these blood
13-16
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levels for severe effects which are not at all a matter of controversy. Were it both logisti-
cal ly feasible to do so on a large scale and were the use of chelants free of health risk to
the subjects, serial provocative chelation testing would appear to be the better indicator of
exposure and risk. Failing this, the only prudent alternative is the use of a large safety
factor applied to blood lead which would translate to an "acceptable" chelatable burden. It
is likely that this blood lead value would lie well below the currently accepted upper limit
of 25 ug/dl (U.S. Centers for Disease Control, 1985), since the safety factor would have to be
large enough to protect against frank plumbism as well as more subtle health effects seen with
non-overt lead intoxication. This rationale from the standpoint of lead toxicokinetics is
also in accord with the growing data base for dose-response relationships of lead's effects on
heme biosynthesis, erythropoiesis, and the nervous system in humans as detailed in Sections
12.3 and 12.4 (see also Section 13.5, below).
Further development and routine use of iji situ mineral tissue testing at time points con-
cordant with on-going exposure and the comparison of such results with simultaneous blood lead
and chelatable lead measurement would be of significant value in further defining what level
of blood lead is indeed an acceptable upper limit.
13.4 DEMOGRAPHIC CORRELATES OF HUMAN LEAD EXPOSURE AND RELATIONSHIPS BETWEEN EXTERNAL AND
INTERNAL LEAD EXPOSURE INDICES
13.4.1 Demographic Correlates of Lead Exposure
Studies of ancient populations using bone and teeth show that levels of internal exposure
of lead today are substantially elevated over past levels. Studies of current populations
living in remote areas far from urbanized cultures show blood lead levels in the range of 1-5
ug/dl. In contrast to the blood lead levels found in remote populations, data from current
U.S. populations generally have geometric means ranging from 10 to 20 ug/dl depending on age,
race, sex, and degree of urbanization. These higher blood lead levels in the United States
appear to be associated with industrialization and widespread commercial use of lead, e.g., in
gasoline combustion.
Age appears to be one of the most important demographic covariates of blood lead levels.
Blood lead levels in children up to six years of age are generally higher than those in non-
occupational ly exposed adults. Children aged two to three years tend to have the highest
levels, as shown in Figure 13-3. Blood lead levels in non-occupationally exposed adults may
increase slightly with age due to skeletal lead accumulation.
Sex has a differential impact on blood lead levels, depending on age. No significant
differences exist between males and females less than seven years of age; males above the age
of seven generally have higher blood lead levels than females. Race also plays a role, in
13-17
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40
35
30
o
25
20
15
.IDAHO STUDY
• NEW YORK SCREENING BLACKS
• NEW YORK SCREENING • WHITES
• NEW YORK SCREENING HISPANICS
NHANES II STUDY • BLACKS
NHANES II STUDY - WHITES
\
0123456789 10
AGE, yr
Figure 13-3. Geometric mean blood lead levels by race and age for younger children in the
NHANES II Study (Annest et al., 1982), the Kellogg Silver Valley, Idaho Study (Yankel et
al., 1977), and the New York Childhood Screening Studies (Billick et al., 1979).
13-18
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that blacks have higher blood lead levels on average than either whites or Hispanics. The
reason for this is not yet fully understood; genetic factors have yet to be fully disentangled
from differential exposure circumstances and other factors.
Blood lead levels also seem to increase with degree of urbanization. Data from NHANES II
show that blood lead levels in the United States, averaged from 1976 to 1980, increase from a
geometric mean of 11.9 ug/dl in rural populations to 12.8 pg/dl in urban populations less than
one million, and increase again to 14.0 ug/dl in urban populations of one million or more.
Recent U.S. blood lead levels show a downward trend occurring consistently across race,
age, and geographic location. This pattern commenced in the early part of the 1970's and has
continued into 1980. The downward trend has occurred from a shift in the entire distribution
and not through a truncation in the high blood lead levels. This consistency suggests a
general causative factor, and attempts have been made to identify the causative element.
Reduction in lead emitted from the combustion of leaded gasoline is a prime candidate (See
discussion under 13.4.2).
Distribution of blood lead levels, examined on a population basis, generally have simi-
larly skewed distributions. That is, blood lead levels from populations thought to be homoge-
nous in terms of demographic and lead exposure characteristics generally follow an approxi-
mately lognormal distribution. Geometric standard deviations (an estimation of dispersion)
for observed distributions from four different studies discussed in Chapter 11 (including
analytic error) are about 1.4 for children and possibly somewhat smaller for adults. This
allows an estimation of the upper tail of the blood lead distribution for the U.S. population,
which would be the population segment expected to be at greater risk.
13.4.2 Relationships Between External and Internal Lead Exposure Indices
There is no question that, across a broad spectrum of external air lead concentrations
ranging upward to beyond 10-20 H9/m3« the relationship between air lead exposures and in-
creases in blood lead levels is nonlinear. However, because one main purpose of this document
is to examine relationships of lead in air and lead in blood under ambient conditions, the
results of studies most appropriate for this purpose were emphasized in Chapter 11. A summary
of the most appropriate studies appears in Table 13-4. At air lead exposures of 3.2 pg/m3 or
less, there is no statistically significant difference between curvilinear and linear blood
lead inhalation relationships. Also, for air lead exposures of 10 jjg/m3 or more, either non-
linear or linear relationships can be fitted. Thus, a reasonably consistent picture emerges
in which the blood lead-air lead relationship by direct inhalation appears to be approximately
linear in the range of normal ambient exposures (0.1-2.0 MO/m3) as discussed in Chapter 7.
Differences among individuals in a given study, and among several studies are large, so that
13-19
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TABLE 13-4. SUMMARY OF BLOOD INHALATION SLOPES (p)
Population Study
Children Angle and
Mclntire (1979),
Omaha, NE
Roels et al.
(1980),
Belgium
Yankel et al.
(1977); Walter
et al. (1980),
Idaho
Adult Males Azar et al.
(1975), five
groups
Griffin et al.
(1975), NY
prisoners
Gross
(1979)
Rabinowitz et
al. (1973,1976,
1977)
Study
Type N
Population 1074
Population 148
Population 879
Population 149
Experiment 43
Experiment 6
Experiment 5
Slope (B),
ug/dl per ug/m3
1.92
2.46
1.52
1.32
1.75
1.25
2.14
Model sensitivity
of slope
(1.40 - 4.40)b
(1.55 - 2.46)b
(1.07 - 1.52)b
(1.08 - 2.39)c
(1.52 - 3.38)e
(1.25 - 1.55)c
(2.14 - 3.51)f
,c,d
,c
,c,d
,d
Selected from among the most plausible statistically equivalent models. For nonlinear
models, slope at 1.0 ug/m3.
Sensitive to choice of other correlated predictors such as dust and soil lead.
cSensitive to linear vs. nonlinear at low air lead.
Sensitive to age as a covariate.
Sensitive to baseline changes in controls.
Sensitive to assumed air lead exposure.
13-20
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pooled estimates of the blood lead inhalation slope depend upon the the weight given to vari-
ous studies. Several studies were selected for analysis, based upon factors described earli-
er. EPA analyses of experimental and clinical studies (Griffin et al., 1975; Rabinovritz et
al., 1973, 1976, 1977; Kehoe 1961a,b,c; Gross 1981; Hammond et al., 1981) suggest that blood
blood lead in adults increases by approximately 1.64 ± 0.22 ug/dl from direct inhalation of
each additional ug/m3 of air lead. EPA anaylsis of Azar's population study (Azar et al.,
1975) yields a slope of 1.32 ± 0.38 for adult males. EPA analyses of other population studies
(Yankel et al., 1977; Roels et al., 1980; Angle and Mclntire, 1979) suggest that, for child-
ren, the blood lead increase is approximately 1.92 (a median estimate) ug/dl per each ug/m3
increment of air lead.
These slope estimates are based on the assumption that an equilibrium level of blood lead
is achieved within a few months after exposure begins. This is only approximately true, since
lead stored in the skeleton may return to blood after some years. Chamberlain et al. (1978)
suggest that long-term inhalation slopes should be about 30 percent larger than these esti-
mates. Inhalation slopes quoted here are associated with a half-life of blood lead in adults
of about 30 days. O'Flaherty et al. (1982) suggest that the blood lead half-life may increase
slightly with duration of exposure, but this has not been confirmed (Kang et al., 1983).
One possible approach would be to regard all inhalation slope studies as equally infor-
mative and to calculate an average slope using reciprocal squared standard error estimates as
weights. This approach has been rejected for two reasons. First, the standard error estima-
tes characterize only the internal precision of an estimated slope, not its representativeness
(i.e., bias) or predictive validity. Secondly, experimental and clinical studies obtain more
information from a single individual than do population studies. Thus, it may not be appro-
priate to combine the two types of studies.
While estimates of the inhalation slope for children are only available from population
studies, the importance of dust ingestion as a non-inhalation pathway for children is estab-
lished by many studies. Slope estimates have been derived for air lead inhalation based on
several such studies (e.g., Angle and Mclntire 1979; Roels et al., 1980; Yankel et al., 1977)
from which the air inhalation and dust ingestion contributions can both be estimated.
Brunekreef (1984) reviewed these and other studies and found wide variations in slope
estimates that include aggregate impacts of direct inhalation and indirect dust contributions.
Such aggregate analyses from some of the better conducted studies yield slope estimates in the
general range of 4-6 ug/dl blood lead per ug/m3 air lead increase. Also, results from another
recent analysis (Angle et al., 1984) suggest that indirect soil/dust contributions contribute
blood lead increases of 4-5 ug/dl in addition to the direct inhalation contribution of 1.92
ug/dl blood lead per ug/m3 air lead.
13-21
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While direct inhalation of air lead is stressed, this is not the only air lead contribu-
tion that needs to be considered. Smelter studies allow partial assessment of the air lead
contributions to soil, dust, and finger lead. Conceptual models allow preliminary estimation
of the propagation of lead through the total food chain as shown in Chapter 7; useful mathe-
matical models to quantify this propagation through the food chain need to be developed. The
direct inhalation relationship does provide useful information on changes in blood lead in
response to changes in air lead on a time scale of several months. However, the indirect
pathways through dust and soil and through the food chain may delay the total blood lead
response to changes in air lead, perhaps by one or more years. The Italian ILE study facili-
tates partial assessment of this delayed response from leaded gasoline as a source.
Dietary absorption of lead varies greatly from one person to another and depends on the
physical and chemical form of the carrier, on nutritional status, and on whether lead is in-
gested with food or between meals. These distinctions are particularly important for consump-
tion by children of leaded paint, dust, and soil. Typical values of 10 percent absorption of
ingested lead into blood have been assumed for adults and 25-50 percent for children.
It is difficult to determine accurate relationships between blood lead levels and lead
levels in food or water. While dietary intake must be estimated by duplicate diets or fecal
lead determinations, water lead levels can be determined with some accuracy. However, the
varying amounts of water consumed by different individuals add to the uncertainty of the esti-
mated relationships.
Quantitative analyses relating blood lead levels and dietary lead exposures have been re-
ported. While studies on infants provide estimates that are in close agreement, only one
individual study is available for adults (Sherlock et al. 1982); another estimate from a num-
ber of pooled studies is also available. These two estimates are in good agreement. Most of
the subjects in the Sherlock et al. (1982) and United Kingdom Central Directorate on Environ-
mental Pollution (1982) studies received quite high dietary lead levels (>300 ug/day). The
fitted cube root equations give high slopes at lower dietary lead levels. On the other hand,
the linear slope of the United Kingdom Central Directorate on Environmental Pollution (1982)
study is probably an underestimate of the slope at lower dietary lead levels. For these
reasons, the Ryu et al. (1983) study appears to be the most reliable, although it only applies
to infants. Estimates for adults can be obtained from the experimental studies, but would
most appropriately apply at high exposure levels (e.g., >300 ug/day). in such studies, most
of the dietary lead intake supplements were so high that many of the subjects had blood lead
concentrations much in excess of 30 ug/dl for a considerable part of the experiment. The
blood lead levels thus may not have completely reflected lead exposure, due to the previously
noted nonlinearity of blood lead response at high exposures. The slope estimates for adult
dietary intake are about 0.02 ug/dl increase in blood lead per ug lead/day total intake, but
13-22
-------
consideration of blood lead kinetics may increase this value to about 0.04. Such values are a
bit lower than those estimated from the population studies extrapolated to typical dietary in-
takes: that is, about 0.05 ug/dl per ug/day. The Ryu et al. (1983) value for infants is much
larger, being about 0.16 ug/dl per ug/day.
The relation between blood lead and water lead is not clearly defined and is often de-
scribed as nonlinear. Water lead intake varies greatly from one person to another. It has
been assumed that children can absorb 25-50 percent of lead in water. Some authors chose to
fit cube root models to their data, although polynomial and logarithmic models were also used.
Unfortunately, the form of the model greatly influences the estimated contributions to blood
lead levels from relatively low water lead concentrations.
Although there is close agreement in the quantitative analyses of the relationship
between blood lead level and dietary lead, there is a larger degree of variability in results
of the various water lead studies. Over a wide range of water lead concentrations, the rela-
tionship is curvilinear, but its exact form has yet to be determined. At typical ambient
water levels for U.S. populations, the relationship appears linear. The only study that
determines the relationship based on lower water lead values (<100 ug/1) is the Pocock et al.
(1983) study. The data from this study, as well as the authors themselves, suggest that the
relationship is linear in this lower range of water lead levels. Furthermore, the estimated
contributions to blood lead levels in this study are quite consistent with the polynomial
models from other studies. For these reasons, the Pocock et al. (1983) slope of 0.06 is con-
sidered to represent the best estimate. The possibility still exists, however, that the
higher estimates of the other studies may be correct in certain situations, especially at
higher water lead levels (>100 ug/1).
Studies relating soil lead to blood lead levels are difficult to compare. The relation-
ship obviously depends on depth of soil lead, sampling method, cleanliness of the home, age of
the children, mouthing activities of the children, and possibly many other factors. Various
soil sampling methods and sampling depths have been used over time, and as such they may not
be directly comparable and may produce a dilution effect of the major lead concentration con-
tribution from dust, which is located primarily in the top 2 cm of the soil. Increases in
soil dust lead significantly increase blood lead in children. From several studies (Yankel et
al., 1977; Angle and Mclntire, 1979) EPA estimates an increase of 0.6-6.8 ug/dl in blood lead
for each increase of 1000 ug/g in soil lead concentration. The values from the Stark et al.
(1982) study of about 2 ug/dl per 1000 ug/g may represent a reasonable median estimate. The
relationship of housedust lead to blood lead is difficult to obtain. Household dust also in-
creases blood lead, as children from the cleanest homes in the Silver Valley/Kellogg Study had
6 ug/dl less lead in blood, on average, than those from the households with the most dust.
13-23
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A number of specific environmental sources of airborne lead have been evaluated for po-
tential direct influence on blood lead levels. Combustion of leaded gasoline appears to be an
extremely important contributor to airborne lead in the United States, as indicated by strong
associations between reductions in nationwide gasoline lead usage and average U.S. blood lead
levels determined by a major population survey on a nationwide level. Studies of data from
blood lead screening programs in specific U.S. metropolitan areas also suggest that the down-
ward trend in blood lead levels noted earlier is due to reductions in air lead levels, mainly
attributable to reductions of lead in gasoline.
In addition, other studies used isotope ratios of lead to estimate the relative propor-
tion of lead in the blood coming from airborne lead or, more specifically, from leaded gaso-
line usuage. For example, from the Isotopic Lead Experiment (ILE) data of Facchetti and Geiss
(1982) and Facchetti (1985), as shown in Table 13-5, the direct inhalation of air lead may
account for 60 percent of the total adult blood lead uptake from leaded gasoline in a large
urban center, but inhalation is a much less important pathway in suburban parts of the region
(19 percent of the total gasoline lead contribution) and in the rural parts of the region (9
percent of the total gasoline lead contribution). EPA analyses of the preliminary results
from the ILE study separated the inhalation and non-inhalation contributions of leaded gaso-
line to blood lead into the following three parts: (1) an increase of about 1.7 ug/dl in
blood lead per (jg/m3 of air lead, attributable to direct inhalation of the combustion products
of leaded gasoline; (2) a sex difference of about 2 ug/dl attributable to lower exposure of
women to indirect (non-inhalation) pathways for gasoline lead; and (3) a non-inhalation back-
ground attributable to indirect gasoline lead pathways, such as ingestion of dust and food,
increasing from about 2 M9/dl in Turin to 3 ug/dl in remote rural areas. The non-inhalation
background represents only two to three years of environmental accumulation at the new experi-
mental lead isotope ratio. It is not clear how to numerically extrapolate these estimates to
subpopulations in the United States; however, it is evident that even in rural and suburban
parts of a metropolitan area, the indirect (non-inhalation) pathways for exposure to leaded
gasoline make a significant contribution to blood lead. This can be seen in Table 13-5. It
should also be noted that the blood lead isotope ratio responded fairly rapidly when the gaso-
line lead isotope ratio returned to its pre-experimental value, but it is not yet possible to
estimate the long-term change in blood lead attributable to persistent exposures to accumu-
lated environmental lead.
The strongest kind of scientific evidence about causal relationships is based on an ex-
periment in which all possible extraneous factors are controlled. The evidence derived from
the Isotopic Lead Experiment comes very close. The experimental intervention consisted of
13-24
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TABLE 13-5. ESTIMATED CONTRIBUTION OF LEADED GASOLINE TO BLOOD LEAD
BY INHALATION AND NON-INHALATION PATHWAYS
Location
Turin
<25 km
>25 km
Air lead
fraction
from
gaso-
line3
0.873
0.587
0.587
Mean
air
lead b
cone. ,
ug/m3
2.0
0.56
0.30
Blood Pb
fraction
from
gaso-
linec
0.214
0.114
0.101
Mean
blood
lead
cone. ,
MQ/dl
21.77
25.06
31.78
Blood
Pb
from
gaso-
line,
Mg/dl
4.66
2.86
3.21
Pb
from
gaso-
line -
in air,
Mg/dl
2.79
0.53
0.28
Non-
inhaled
Pb from
gaso-
line,
ug/dl
1.80
2.33
2.93
Estimated
fraction
gas-lead
inhala-
tion"
0.60
0.19
0.09
Fraction of air lead in Phase 2 attributable to lead in gasoline.
Mean air lead in Phase 2, ug/m3.
cMean fraction of blood lead in Phase 2 attributable to lead in gasoline.
Mean blood lead concentration in Phase 2, ug/dl.
eEstimated blood lead from gasoline = (c) x (d).
Estimated blood lead from gas inhalation = B x (a) x (b), B = 1.6.
^Estimated blood lead from gas, non-inhalation = (f)-(e).
Fraction of blood lead uptake from gasoline attributable to direct inhalation = (f)/(e).
Data: Facchetti and Geiss (1982); Facchetti (1985).
replacing the normal 206Pb/207Pb isotope ratio by a very different ratio. There is no plausi-
ble mechanism by which other concurrent lead exposure variables (food, water, beverages,
paint, industrial emissions) could have also changed their isotope ratios. Hence the very
large changes in isotope ratios in blood were responding to the change in gasoline. Our
analyses (Chapter 11) show that consideration of inhalation of community air lead alone pro-
bably substantially underestimates the total effect of gasoline lead, at least in the 35 sub-
jects whose blood leads were tracked in the ILE Preliminary Study. Spengler et al. (1984), as
discussed in Section 11.3, also suggest that part of the extra exposure could possibly be
attributed to exposure to higher-than-ambient air lead concentrations inside motor vehicles,
e.g., on the trip to work; however, no data are presently available to confirm this hypothe-
sis.
Primary lead smelters, secondary lead smelters, and battery plants emit lead directly in-
to the air and ultimately increase soil and dust lead concentrations in their vicinity.
Adults, and especially children, have been shown to exhibit elevated blood lead levels when
living close to these sources. Blood lead levels in these residents have been shown to be
13-25
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related to air lead, as well as to soil or dust lead exposures. In addition, individuals
(especially children) living in housing units with deteriorating or weathering lead-based
paint may also be exposed via lead accumulated in dust or soils within or around their dwell-
ings.
13.4.3 Proportional Contributions of Lead in Various Media to Blood Lead
in Human Populations
The various mathematical descriptions of the relationship of blood lead to lead in indi-
vidual media—air, food, water, dust, soil—were discussed in some detail in Chapter 11 and
concisely in the preceding section (13.4.2) of this chapter. Using values for lead intake/
content of these media which appear to represent the current exposure picture for human popu-
lations in the United States, these relationships are further employed in this section to
estimate proportional inputs to total blood lead levels in U.S. children. Such an exercise is
of help in providing an overall perspective on which routes of exposure are of most signifi-
cance in terms of contributions to blood lead levels seen especially in urban children, the
population group in the United States at greatest risk for lead exposure and its toxic ef-
fects.
Table 13-6 tabulates the relative direct contributions of air lead to blood lead at dif-
ferent air lead levels for calculated typical background levels of lead from food, water, and
dust for children in the United States. Also listed are the direct and indirect contributions
of air lead to blood lead at varying air lead levels for children, given calculated typical
background levels of blood lead. Calculations and assumptions used in deriving the estimates
shown in Table 13-6 are summarized in footnotes to that table. The diet contributions listed
in the table, for example, are based on the following: (1) estimated average background
levels of lead (from non-air and air sources) in food ingested per day by children, as
delineated in Table 7-19; and (2) the value of 0.16 ug/dl of blood increase per ug/day food
lead intake found by Ryu et al. (1983) for infants. Similarly, values for other parameters
used in Table 13-6 are obtained from work discussed in Chapters 7 and 11.
It is of interest to compare (1) estimated blood lead values predicted in Table 13-6 to
occur at particular air lead concentrations with (2) actual blood lead levels observed for
children living in the United States in areas with comparable ambient air concentrations. As
an example, NHANES II survey results for children living in rural areas and urban areas of
more than one million population or less than one million were presented in Table 11-5. For
children (aged 0.5-5 yr) living in urban areas of >1 million, the mean blood lead value was
16.8 ug/dl, a value representative of average blood lead levels nationwide for preschool chil-
dren living in large urban areas during the NHANES survey period (1976 to February, 1980).
13-26
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Ambient air lead concentrations (quarterly averages) during the same time period (1976-1979)
for a geographically diverse sample of large urban areas in the United States (population
>1 million) 9^ available from Table 7-3. The air lead levels during 1976-1979 averaged 1.06
pg/m3 for all cities listed in Table 7-3 and 1.12 ug/m3 for eight cities in the table that
were included in the NHANES II study (i.e., Boston, New York, Philadelphia, Detroit, Chicago,
Houston, Los Angeles, and Washington, DC). The Table 13-6 blood lead values of 12.6-14.6
ug/dl estimated for air lead levels of 1.0-1.25 ug/m3 approximate the observed NHANES II
average of 16.8 ug/dl for children in large urban areas with average air lead levels of
1.06-1.12 ug/m3. The NHANES II blood lead values for preschool children would be expected to
be somewhat higher than the estimates in Table 13-6 because the latter were derived from FDA
data for 1981-1983, which were lower than the FDA values for the 1976-1980 NHANES II period
(see Chapter 7). FDA data for food, water, and beverages for the 1976-1980 period are not in
a form exactly comparable to the 1981-1983 data used in calculating background contributions
1n Table 13-6, but do suggest that lead levels in those media declined by about 20 percent
from the 1976-1980 period to 1981-1983. If background contributions in Table 13-6 were cor-
rected (i.e., increased by 20 percent) to be comparable to the 1976-1980 period, then the
blood lead levels of children exposed to 1.25 ug/m3 air lead would increase to 15.5 ug/dl, a
value even closer to the mean of 16.8 ug/dl found for NHANES II children living in urban
environments (>1 million) during 1976-1980.
13.5 BIOLOGICAL EFFECTS OF LEAD RELEVANT TO THE GENERAL HUMAN POPULATION
13.5.1 Introduction
It is clear from the wealth of available literature reviewed in Chapter 12 that there
exists a continuum of biological effects associated with lead across a broad range of expo-
sure. At rather low levels of lead exposure, biochemical changes, e.g., disruption of certain
enzymatic activities involved in heme biosynthesis and erythropoietic pyrimidine metabolism,
are detectable. Heme biosynthesis is a generalized process in mammalian species, including
man, with importance for normal physiological functioning of virtually all organ systems.
With increasing lead exposure, there are sequentially more intense effects on heme synthesis
as well as a broadening of effects to additional biochemical and physiological mechanisms in
various tissues. In addition to heme biosynthesis impairment at relatively low levels of lead
exposure, disruption of normal functioning of the erythropoietic and nervous systems are among
the earliest effects observed as a function of increasing lead exposure. With increasingly
Intense exposure, more severe disruption of the erythropoietic and nervous systems occur and
additional organ systems are affected, resulting, for example, in manifestation of renal
13-27
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TABLE 13-6. CONTRIBUTIONS FROM VARIOUS MEDIA TO BLOOD LEAD LEVELS (ug/dl) OF
U.S. CHILDREN (AGE = 2 YEARS): BACKGROUND LEVELS AND INCREMENTAL CONTRIBUTIONS FROM AIR
Air lead, ug/m3
Source
Background-non air
Food, Water and
Beverages
Subtotal
0
2.37
0.30
O7
0.25
2.37
0.30
2.67
0.50
2.37
0.30
2767
0.75
2.37
0.30
2.67
1.00
2.37
0.30
2.67
1.25
2.37
0.30
2767
1.50
2.37
0.30
2T67
Background-air
Food, Water and
Beverages0 1.65 1.65 1.65 1.65 1.65 1.65 1.65
deposited
Inhaled air
Total
from air)
e
0.00
0.00
4.32
1.57
0.50
6.39
3.09
1.00
8.41
4.
1.
10.
70
50
52
6.
2.
12.
27
00
59
7.
2.
14.
84
50
66
9.40
3.00
16.72
aFrom Table 7-19, (25.1 - 10.3) ug/day x (0.16 from Ryu et al., 1983) = 2.37 ug/dl.
From Chapter 7, 1/10 dust not atmospheric. Using Angle et al. (1984) low area (Area S)
for soil and house dust and their regression equation, we have: (1/10) x (97 ug/g x
0.00681 + 324 ug/g x 0.00718) = 0.30 ug/dl. Alternatively, the consumption from non air
would be (1/10) x (97 ug/g soil dust + 324 ug/g house dust) x 0.05 grains ingested of
each = 2.1 ug ingested. Using Ryu et al. (1983), 2.1 x 0.16 = 0.34 ug/dl added to blood.
cAs in (a) above, but using 10.3 instead of (25.1 - 10.3) yields 1.67 ug/dl. Values are
derived for component of background Pb in food from past deposition from air onto soil and
into other media leading into human food chain (not expected to change much except over
long-term).
The regression equations of Angle et al. (1984) are used, as well as levels of soil dust
and house dust in the low area (S) and high area (C) of that study. For example, the
increase at 1.0 ug/m3 in air would result in increases in soil as follows:
Oias "- 0!29 x (519-97) = 526 ug/g
Similarly the increase in house dust would be:
(625 - 324) = 374 ug/g
The effect on blood lead would be (526 x 0.00681) + (374 x 0.00718) = 6.27 ug/dl .
eUsing the 2.0 slope from Angle et al. (1984), i.e., 1.93 rounded up.
13-28
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effects, disruption of reproductive functions, and impairment of immunological functions. At
sufficiently high levels of exposure, the damage to the nervous system and other effects can
be severe enough to result in death or, in some cases of non-fatal lead poisoning, long-
lasting sequelae such as permanent mental retardation.
As discussed in Chapter 12 of this document, numerous new studies, reviews, and critiques
concerning lead-related health effects have been published since the issuance of the earlier
EPA Lead Criteria Document in 1977. Of particular importance for present criteria development
purposes are those new findings, taken together with information available at the writing of
the 1977 Lead Criteria Document, which have bearing on the establishment of quantitative
dose-effect or dose-response relationships which can be potentially viewed as adverse health
effects likely to occur among the general population at or near existing ambient air concen-
trations of lead in the United States. Key information regarding observed health effects
and their implications are discussed below for adults and children.
For the latter group, children, emphasis is placed on the discussion of (1) heme biosyn-
thesis effects, (2) certain other biochemical and hematological effects, and (3) the disrup-
tion of nervous system functions. All of these appear to be among those effects of most con-
cern for potential occurrence in association with exposure to existing U.S. ambient air lead
levels for the population group at greatest risk for lead-induced health effects (i.e., chil-
dren £6 years old). Emphasis is also placed on the delineation of internal lead exposure
levels, as defined mainly by blood lead (PbB) levels likely associated with the occurrence of
such effects. Also discussed are characteristics of the subject effects that are of crucial
importance with regard to the determination of which might reasonably be viewed as constitu-
ting "adverse health effects" in affected human populations.
Over the years, there have been superimposed on the continuum of lead-induced biological
effects various judgments as to which specific effects observed in man constitute "adverse
health effects." Such judgments involve not only medical consensus regarding the health sig-
nificance of particular effects and their clinical management, but also incorporate societal
value judgments. Such societal value judgments often vary depending upon the specific overall
contexts in which they are applied; e.g., in judging permissible exposure levels for occupa-
tional versus general population exposures to lead. For some lead exposure effects, e.g.,
severe nervous system damage resulting in death or serious medical sequelae consequent to in-
tense lead exposure, there exists little or no disagreement as to these being significant
"adverse health effects." For many other effects detectable at sequentially lower levels of
lead exposure, however, the demarcation lines as to which effects represent adverse health ef-
fects and the lead exposure levels at which they are accepted as occurring are neither sharp
13-29
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nor fixed, having changed markedly during the past several decades. That is, from an histori-
cal perspective, levels of lead exposure deemed to be acceptable for either occupationally-
exposed persons or the general population have been steadily revised downward as more sophis-
ticated biomedical techniques have revealed formerly unrecognized biological effects and con-
cern has increased in regard to the medical and social significance of such effects. As a
concrete example, pediatric blood lead concentrations deemed to be associated with unaccept-
able risk of lead toxicity have been repeatedly revised downward by the U.S. Public Health
Service (see CDC, 1985).
It is difficult to provide a definitive statement of all criteria by which specific bio-
logical effects associated with any given agent can be judged to be "adverse health effects."
Nevertheless, several criteria are currently well-accepted as helping to define which effects
should be viewed as "adverse." These include the following: (1) impaired normal functioning
of a specific tissue or organ system itself; (2) reduced reserve capacity of that tissue or
organ system in dealing with stress due to other causative agents; (3) the reversibility/
irreversibility of the particular effect(s); (4) the relative frequency of a given effect; (5)
presence of the effect in a vulnerable segment of the population; and (6) the cumulative or
aggregate impact of various effects on individual organ systems on the overall functioning and
well-being of the individual.
Examples of possible uses of such criteria in evaluating lead effects can be cited for
illustrative purposes. For example, impairment of heme synthesis intensifies with increasing
lead exposure until hemoprotein synthesis is inhibited in many organ systems, leading to re-
ductions in such functions as oxygen transport, cellular energetics, neurotransmitter func-
tions, detoxification of xenobiotic agents, and biosynthesis of important substances such as
1,25-dihydroxyvitamin D. In Figure 13-4, the far-ranging impact of lead on the body heme pool
and associated disruption of many physiological processes is depicted, based on data discussed
in Sections 12.2 and 12.3. Furthermore, inspection of Figure 13-4 reveals effects that can be
viewed as intrinsically adverse as well as those that reduce the body's ability to cope with
other forms of toxic stress, e.g., reduced hepatic detoxification of many types of xenobiotics
and, possibly, impairment of the immune system. The liver effect can also be cited as an
example of reduced reserve capacity pertinent to consideration of the effects of lead, as the
reduced capacity of the liver to detoxify certain drugs or other xenobiotic agents results
from lead effects on hepatic detoxification enzyme systems.
In regard to the issue of reversibility/irreversibility of lead effects, there are really
two dimensions to the issue that need to be considered, i.e.: (1) biological reversibility or
irreversibility characteristic of the particular effect in a given organism; and (2) the gene-
rally less-recognized concept of exposure reversibility or irreversibility. Severe central
13-30
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REDUCTION OF
HCMt BODY POOL
RYTHROPOIETIC REDUCED AIIEMIA - REDUCED
SYNTHESIS TO ALL TISSUES
EXACERBATION OF
OTHER STRESS AGENTS
/
EFFECTS ON NEURONS
AXONS. AND
SCHWANN CELLS
/ *
NEURAL REDUCED HEMOFROTElNS IMPAIRED j
EFIICTI "" IM..CYTOCHROMESI ~*~ IHIULAR h-»-
ENERGETICS \
\
DISTURBED IMUNO
RECULATORVRDLE
41 OF CALCIUM 4
1
RENAL ENDOCRINE^ R[DUCED I.!S (OKI, - / ^ DISTUHIED CALCIUM / ^
EFFICTI VITAMIN 0 \ METABOLISM \~
» \
IMPAIRED MVELINATION
AND NERVE CONDUCTION
IMPAIRED DEVELOPMENT
OF NERVOUS SYSTEM
TISSUE HONEOSTASIS
IMPAIRED CALCIUM
ROLE AS SECOND
MESSENGER
\
* DISTURBED ROLE IN *
TUMORICENESIS
CONTROL
X
1 IMPAIRED /
J DETOXIFICATION /
^| OF IENOBIOTICS \
/ \
MIPAT.C __j ""UCEOHEMEFOR ]/
EFFECTS H HEMERECULAIEU I
\ TRANSFORMATIONS |\
"~" ~~~ \ . /
VI IMPAIRED METABOLISM /
OFENDOCiNOUS V
AGONISTS [\
IMPAIRED CALCIUM
ROLE IN CYCLIC
NUCLEOTIDE METABOLISM
IMPAIRED DETOXIFICATION
rOF ENVIRONMENTAL
TOXINS
CARDIOVASCULAR
OTHER HVPOXIC EFFECTS
TOOTH DEVELOPMENT
]
IMPAIRED
DETOXIFICATION
OF DRUGS
I ALTERED METABC: ISM
1 OF TRYPTOPHAN
ELEVATED BRAIN
-•M LEVELS OF TRYPTOPHAN,
[ SEROTONIN. ANOHIAA
t
IMPAIRED DISTURBED INOOLEAMINE
J HVDROXYLATION NEUROTRANSMITTER
1 OF COKIISOl | FUNCTION
Figure 13-4. Multi-organ impact of reductions of heme body pool by lead. Impairment of home
synthesis by lead (see Section 12.3) results in disruption of a wide variety of important physio-
logical processes in many organs and tissues. Particularly well documented are erythropoietic,
neural, renal-endocrine, and hepatic effects indicated above by solid arrows ( »-). Plausible
further consequences of heme synthesis interference by lead which remain to be more conclu-
sively established are indicated by dashed arrows ( •*»).
13-31
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nervous system damage resulting from Intense, high-level lead exposure Is generally accepted
as an irreversible effect of lead exposure; the reversibility/irreversibility of certain more
difficult-to-detect neurological effects occurring at lower lead exposure levels, however,
remains a matter of some controversy. The concept of exposure reversibility/irreversibility
can be illustrated by the case of urban children of low socioecomomic status showing disturb-
ances in heme biosynthesis and erythropoiesis. Biologically, these various effects may be
considered reversible; the extent to which actual reversibility occurs, however, is determined
by the feasibility of removing these subjects from their particular lead exposure setting. If
such removal from exposure is unlikely or does not occur, then such effects will logically
persist and, defacto, constitute essentially irreversible effects.
The issues of frequency of effects and vulnerable segments of the population in whom
these effects occur are intimately related. As detailed later in Section 13.7, young chil-
dren—particularly inner-city children—constitute a high risk group because they do show a
high frequency of certain health effects, as summarized below.
13.5.2 Dose-Effect Relationships for Lead-Induced Health Effects
13.5.2.1 Human Adults. The lowest observed effect levels (in terms of blood lead concentra-
tions) thus far credibly associated with particular health effects of concern for human adults
in relation to specific organ systems or generalized physiological processes, e.g., heme syn-
thesis, are summarized in Table 13-7. That table should be viewed as representing lowest blood
lead levels thus far credibly associated with unacceptable risk for a given effect occurring
among at least some adults. As such, many other individuals may not experience the stated
effect until distinctly higher blood lead levels are reached, due to wide ranges of individual
biological susceptibility, variations in nutritional status, and other factors.
The most serious effects associated with markedly elevated blood lead levels are severe
neurotoxic effects that include irreversible brain damage, as indexed by the occurrence of
acute or chronic encephalopathic symptoms observed in both humans and experimental animals.
For most human adults, such damage typically does not occur until blood lead levels exceed
100-120 ug/dl. Often associated with encephalopathic symptoms at these or higher blood lead
levels are severe gastrointestinal symptoms and objective signs of effects on several other
organ systems. Precise threshold(s) for occurrence of overt neurological and gastrointestinal
signs and symptoms of lead exposure in cases of subencephalopathic lead intoxication remain to
be established, but such effects have been observed in adult lead workers at blood lead levels
as low as 40-60 ug/dl, notably lower than levels earlier thought to be "safe" for adult lead
exposure. Other types of health effects occur coincident with the above overt neurological
and gastrointestinal symptoms indicative of marked lead intoxication. These range from frank
peripheral neuropathies to chronic nephropathy and anemia.
13-32
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TABLE 13-7. SUMMARY OF LOWEST OBSERVED EFFECT LEVELS FOR KEY LEAD-INDUCED HEALTH EFFECTS IN ADULTS
Lowest
effect
100-120
80
60
50
co
30
25-30
15-20
<10
observed *
level (PbB)
ug/dl
ug/dl
ug/dl
ng/di
ug/dl
ug/dl
Mg/di
ug/dl
ug/dl
Heme synthesis and
hematological effects
Frank anemia
Reduced hemoglobin
production
Increased urinary ALA and
elevated coproporphyrins
Erythrocyte protoporphyrin
(EP) elevation in males
Erythrocyte protoporphyrin
(EP) elevation in females
ALA-D inhibition
Neurological Effects on Reproductive Cardiovascular
effects the kidney function effects effects
Encephalopathic signs Chronic
and symptoms nephropathy
-j- 1
1
Overt subencephalopathic '
neurological symptoms
*
Peripheral nerve dysfunction - -
(slowed nerve conduction)
.1.
:emale reproductive
effects
Utered testicular
function
-
Elevated blood
pressure
(White males J
aged 40-59
! i
?
PbB = blood lead concentrations.
Source: This report.
-------
Toward the lower range of blood lead levels associated with overt lead intoxication or
somewhat below, less severe but important signs of impairment in normal physiological func-
tioning in several organ systems are evident among apparently asymptomatic lead-exposed
adults, including the following: (1) slowed nerve conduction velocities indicative of peri-
pheral nerve dysfunction (at levels as low as 30-40 ug/dl); (2) altered testicular function
(at 40-50 ug/dl); and (3) reduced hemoglobin production (at approximately 50 ug/dl) and other
signs of impaired heme synthesis evident at still lower blood lead levels. All of these ef-
fects point toward a generalized impairment of normal physiological functioning across several
different organ systems, which becomes abundantly evident as adult blood lead levels exceed
30-40 ug/dl. Evidence for impaired heme synthesis effects in blood cells exists at still
lower blood lead levels in adults, as does evidence for elevated blood pressure in middle-aged
white males (aged 40-59). The significance of impaired heme synthesis effects and evidence of
impairment of other biochemical processes important in cellular energetics are discussed below
in relation to children.
13.5.2.2 Children. Table 13-8 summarizes lowest observed effect levels for a variety of im-
portant health effects observed in children. Again, as for adults, it can be seen that lead
impacts many different organ systems and biochemical/physiological processes across a wide
range of exposure levels. Also, again, the most serious of these effects is the severe, irre-
versible central nervous system damage manifested in terms of encephalopathic signs and symp-
toms. In children, effective blood lead levels for producing encephalopathy or death are
lower than for adults, starting at approximately 80-100 ug/dl. Permanent severe mental retar-
dation and other marked neurological deficits are among lasting neurological sequelae typical-
ly seen in cases of non-fatal childhood lead encephalopathy. Other overt neurological signs
and symptoms of subencephalopathic lead intoxication are evident in children at lower blood
lead levels (e.g., peripheral neuropathies detected in some children at levels as low as 40-60
ug/dl). Chronic nephropathy, indexed by aminoaciduria, is most evident at high exposure
levels over 100 pg/dl, but may also exist at lower levels (e.g., 70-80 ug/dl). In addition,
colic and other overt gastrointestinal symptoms clearly occur at similar or still lower blood
lead levels in children, at least down to 60 pg/dl. Frank anemia is also evident by 70 ug/dl,
representing an extreme manifestation of the reduced hemoglobin synthesis observed at blood
lead levels as low as 40 ug/dl, along with other signs of marked inhibition of heme synthesis
at that exposure level. All of these effects are reflective of the widespread marked impact
of lead on the normal physiological functioning of many different organ systems and some are
evident in children at blood lead levels as low as 40 ug/dl; and all of them are widely accep-
ted as being clearly adverse health effects.
Additional studies demonstrate evidence for further, important health effects occurring
in non-overtly lead-intoxicated children at similar or lower blood lead levels than those
13-34
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TABLE 13-8. SUMMARY OF LOWEST OBSERVED EFFECT LEVELS FOR KEV LEAD-INDUCED HEALTH EFFECTS IN CHILDREN
CO
I
Lowest observed
effect level (PbB)*
80-100 ug/dl
70 pg/dl
60 ug/dl
50 ug/dl
40 pg/dl
Heme synthesis and
hematological effects
Frank anemia
Reduced hemoglobin
Neurological
effects
Encephalopathic
signs and symptoms
Peripheral neuropathies
Peripheral nerve dysfunction
Renal system
effects
Chronic nephropathy
(aminoaciduria, etc.}
Gastrointestinal
effects
Colic, other overt
gastrointestinal symptoms
.!.
30 pg/dl
15 ug/dl
10 ug/dl
synthesis
Elevated coproporphyrin
Increased urinary ALA
Erythrocyte protooorphyin
elevation
AlA-D inhibition
Py-5-Mt activity
inhibition
(slowed NCVs)
CHS cognitive effects
(IQ deficits, etc.)
Altered CMS electrophysiological
responses
Vitamin D metabolism
interference
*Pbfl = blood lead concentrations.
tPy-5-N = pyri«idine-5'-nucleot1dase.
Source: This report.
-------
indicated above for overt intoxication effects. Among the most important of the effects dis-
cussed in Chapter 12 are neuropsychological and electrophysiological effects evaluated as
being associated with low-level lead exposures in non-overtly lead-intoxicated children.
Indications of peripheral nerve dysfunction, indexed by slowed nerve conduction velocities
(NCV), have been shown in children down to blood lead levels as low as 30 ug/dl. AS for CNS
effects, none of the available studies on the subject, individually, can be said to prove con-
clusively that significant cognitive (IQ) or behavioral effects occur in children at blood
lead levels <30 ug/dl. However, the most recent neurobehavioral studies of CNS cognitive (IQ)
effects collectively demonstrate associations between neuropsychologic deficits and low-level
lead exposures in young children resulting in blood lead levels ranging to below 30 ug/dl
The magnitudes of average observed IQ deficits generally appear to be approxiamtely 5 points
at mean blood lead levels of 50-70 pg/dl, about 4 points at mean blood lead levels of 30-50
pg/dl, and 1-2 points at mean blood lead levels of 15-30 M9/dl. Somewhat larger decrements
have been reported for the latter blood lead range among children of lower socioeconomic
status families.
Additional recent studies have obtained results at blood lead values mainly in the 15-30
ug/dl range indicative of small, but not unimportant, effects of lead on the ability to focus
attention, appropriate social behavior, and other types of behavioral performance. However
due to specific methodological problems with each of these various studies (as noted in Chap-
ter 12), much caution is warranted that precludes conclusive acceptance of the observed
effects being due to lead rather than other (at times uncontrolled for) potentially confound-
ing variables. This caution is particularly warranted in view of other well-conducted studies
that have appeared in the literature which did not find statistically significant associations
between lead and similar effects at blood lead levels below 30 ug/dl. Still, because such
latter studies even found some small effects remaining after correction for confounding
factors, lead cannot be ruled out as an etiological factor contributing to the induction of
such effects in the 15-30 ug/dl range, based on existing published studies.
Also of considerable importance are studies which provide evidence of changes in EEC
brain wave patterns and CNS evoked potential responses in non-overtly lead intoxicated chil-
dren. The work of Burchfiel et al. (1980) indicates significant associations between IQ de-
crements, EEG pattern changes, and lead exposures among children with average blood lead
levels falling in the range of 30-50 vg/tf. Research results provided by Otto et al. (1981,
1982, 1983) also demonstrate clear, statistically significant associations between electrophy-
siological (SW voltage) changes and blood lead levels in the range of 30-55 ug/dl and analo-
gous associations at blood lead levels below 30 pg/dl (with no evident threshold down to 15
ug/dl or somewhat lower). In this case, the presence of electrophysiological changes observed
13-36
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upon follow-up of some of the same children two years and five years later suggests persis-
tence of such effects even in the face of later declines in blood lead levels and, therefore,
possible long-term persistence of the observed electrophysiological CMS changes. However, the
reported electrophysiological effects in this case were not found to be significantly associ-
ated with IQ decrements.
While the precise medical or health significance of the neuropsychological and electro-
physiological effects found by the above studies to be associated with low-level lead expo-
sures is difficult to fully define at this time, the IQ deficits and other behavioral changes
likely impact the intellectual development, school performance, and social development of the
affected children sufficiently so as to be regarded as adverse. This is especially true if
such impaired intellectual development or school performance and disrupted social development
are reflective of persisting, long-term effects of low-level lead exposure in early childhood.
The issue of persistence of such lead effects still remains to be more clearly resolved, with
some study results reviewed in Chapter 12 and mentioned above suggesting relatively short-
lived or markedly decreasing lead effects on neuropsychological functions over a few years
from early to later childhood and other studies suggesting that significant low-level lead-
induced neurobehavioral and EEC effects may, in fact, persist into later childhood. Despite
any remaining ambiguities of the above type, however, the medical community has highlighted
(CDC, 1985) lead-induced neurobehavioral effects (e.g., IQ deficits and other neuropsychologic
effects) as one basis for viewing pediatric blood lead levels below 25-30 ug/dl as being asso-
ciated with unacceptable risk for lead-induced toxicity.
In regard to additional studies reviewed in Chapter 12 concerning the neurotoxicity of
lead, certain evidence exists which suggests that neurotoxic effects may be associated with
lead-induced alterations in heme synthesis, resulting in an accumulation of ALA in brain which
affects CNS GABA synthesis, binding, and/or inactivation by neuronal reuptake after synaptic
release. Also, available experimental data suggest that these effects may have functional
significance in the terms of this constituting one mechanism by which lead may increase the
sensitivity of rats to drug-induced seizures and, possibly, by which GABA-related behavioral
or physiological control functions are disrupted. Unfortunately, the available research data
do not allow credible direct estimates of blood lead levels at which such effects might occur
in rats, other non-human mammalian species, or man. Inferentially, however, one can state
that threshold levels for any marked lead-induced ALA impact on CNS GABA mechanisms are most
probably at least as high as blood lead levels at which significant accumulations of ALA have
been detected in erythrocytes or non-blood soft tissues (see below). Regardless of any dose-
effect levels inferred, though, the functional and/or medical significance of lead-induced ALA
effects on CNS mechanisms at low levels of lead exposure remains to be more fully determined
and cannot, at this time, be unequivocably seen as an adverse health effect.
13-37
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Research concerning lead-induced effects on heme synthesis also provides information of
importance in evaluating what blood lead levels are associated with significant health effects
in children. As discussed earlier, in Chapter 12 and Section 13.4, lead affects heme synthe-
sis at several points in its metabolic pathway, with consequent impact on the normal function-
ing of many body tissues. The activity of the enzyme ALA-S, catalyzing the rate-limiting step
of heme synthesis, does not appear to be significantly affected until blood lead levels reach
or exceed approximately 40 ug/dl. The enzyme ALA-D, which catalizes the conversion of ALA to
porphobilinogen as a further step in the heme biosynthetic pathway, appears to be affected at
much lower blood lead levels as indexed directly by observations of ALA-D inhibition or in-
directly in terms of consequent accumulations of ALA in blood and non-blood tissues. More
specifically, inhibition of erythrocyte ALA-D activity has been observed in humans and other
mammalian species at blood lead levels even below 10-15 ug/dl, with no clear threshold evi-
dent. Correlations between erythrocyte and hepatic ALA-D activity inhibition in lead workers
at blood lead levels in the range of 12-56 ug/dl suggest that ALA-D activity in soft tissues
(e.g., brain, liver, kidney, etc.) may be inhibited at similar blood lead levels at which ery-
throcyte ALA-D activity inhibition occurs, resulting in accumulations of ALA in both blood and
soft tissues.
Some studies indicate that increases in both blood and urinary ALA occur below the cur-
rent commonly-accepted blood lead level of 40 ug/dl. Such increases in blood and urinary ALA
are detectable in humans at blood lead levels below 30 ug/dl, with no clear threshold evident
down to 15-20 ug/dl, although other data exist which fail to show any relationship below 40
ug/dl blood lead. Other studies have demonstrated significant elevations in rat brain,
spleen, and kidney ALA levels consequent to acute or chronic lead exposure, but no clear blood
lead levels can yet be specified at which such non-blood tissue ALA increases occur in humans.
It is reasonable to assume, however, that ALA increases in non-blood tissues likely begin to
occur at roughly the same blood lead levels associated with increases in erythrocyte ALA
levels.
Lead also affects heme synthesis beyond metabolic steps involving ALA, leading to the
accumulation of porphyrin in erythrocytes as the result of impaired iron insertion into the
porphyrin moiety to form heme. The porphyrin acquires a zinc ion in lieu of the native iron,
and the resulting accumulation of blood zinc protoporphyrin (ZPP) tightly bound to erythro-
cytes for their entire life (120 days) represents a commonly employed index of lead exposure
for medical screening purposes. The threshold for elevation of erythrocyte protoporphyrin
(EP) levels is well-established as being 25-30 ug/dl in adults and approximately 15 ug/dl for
young children, with significant EP elevations (>l-2 standard deviations above reference nor-
mal EP mean levels) occurring in 50 percent of all children studied as blood lead approaches
or moderately exceeds 30 ug/dl.
13-38
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Medically, small increases in EP levels were previously not viewed as being of great
concern at initial detection levels around 15-20 ug/dl in children. However, EP increases
become more worrisome when markedly greater, significant elevations occur as blood lead levels
reach 20 to 30 ug/dl and additional signs of significantly deranged heme synthesis begin to
appear, along with indications of functional disruption of various organ systems. Previously,
such other signs of significant organ system functional disruptions had only been credibly
detected at blood lead levels distinctly in excess of 30 ug/dl, e.g., inhibition of hemoglobin
synthesis starting at 40 ug/dl and significant nervous system effects at 50-60 ug/dl. This
served as a basis for CDC's 1978 statement establishing 30 ug/dl blood lead as a criteria
level for undue lead exposure for young children. At the present time, however, the medical
community (CDC, 1985) accepts EP elevations associated with PbB levels of 25 ug/dl or higher
as being unacceptable in pediatric populations.
Recently, it has also been demonstrated in children that lead is negatively correlated
with circulating levels of the vitamin D hormone, 1,25-dihydroxyvitamin D, with the negative
association existing down to 12 ug/dl of blood lead. This effect of lead is of considerable
significance on two counts: (1) altered levels of l,25-(OH)2-vitamin D not only impact cal-
cium homeostasis (affecting mineral metabolism, calcium as a second messenger, and calcium as
a mediator of cyclic nucleotide metabolism) but also likely impact its known role in immuno-
regulation and mediation of tumorigenesis; and (2) the effect of lead on l,25-(OH)2-vitamin D
is a particularly robust one, with blood lead levels of 30-50 ug/dl resulting in decreases in
the hormone that overlap comparable degrees of decrease seen in severe kidney injury or cer-
tain genetic diseases.
Erythrocyte Py-5-N activity in children has also been demonstrated to be negatively im-
pacted by lead at exposures resulting in blood lead levels markedly below 30 ug/dl (i.e., to
levels below 5 ug/dl with no evident threshold). Extensive reserve capacity exists for this
blood enzyme, such that it is not markedly depleted until blood lead levels reach approximate-
ly 30-40 ug/dl, arguing for the Py-5-N effect in and of itself as perhaps not being particu-
larly adverse until such blood lead levels are reached. However, the observation of Py-5-N
inhibition is more arguably indicative of more widespread impacts on pyrimidine metabolism in
general in additional organs and tissues besides blood, such that lead exposures lower than 30
ug/dl resulting in measurable Py-5-N inhibition in erythrocytes may be of greater medical con-
cern when viewed from this broader perspective.
Also adding to the concern about relatively low exposure levels of lead are the results
of an expanding array of animal toxicology studies which demonstrate the following: (1) per-
sistence of lead-induced neurobehavioral alterations well into adulthood long after
termination of perinatal lead exposure early in development of several mammalian species;
13-39
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(2) evidence for uptake and retention of lead in neural and non-neuronal elements of the CNS,
including long-term persistence in brain tissues after termination of external lead exposure
and blood lead levels have returned to "normal"; and (3) evidence from various in v ivo and \n
vitro studies indicating that, at least on a subcellular-molecular level, no threshold may
exist for certain neurochemical effects of lead.
Given the above new evidence that is now available, indicative of significant lead ef-
fects on nervous system functioning and other important physiological processes as blood lead
levels increase above 15-20 ug/dl and reach 20 to 30 ug/dl, the rationale for considering 30
pg/dl as a "maximum safe" blood lead level (as was the case in setting the 1978 EPA lead
NAAQS) was called into question and substantial impetus provided for revising the criteria
level downward. At this time, it is difficult to identify specifically what blood lead
criteria level would be appropriate in view of the existing medical information. Clearly,
however, 30 ug/dl does not afford any margin of safety before blood lead levels are reached
that are associated with unacceptable risk of notable adverse health effects occurring in some
children. This is based on at least two grounds: (1) blood lead levels in the 30-40 ug/dl
range are now known to "mask", for some children, markedly elevated chelatable body lead
burdens that are comparable to lead burdens seen in other children displaying overt signs and
symptoms of lead intoxication and (2) blood lead levels in the 30-40 ug/dl range are also
associated with the onset of deleterious effects in several organ systems which are either
individually or collectively seen as being adverse. These and other considerations have led
the medical community (CDC, 1985) to define 25 ug/dl PbB as a level associated with unaccept-
able risk for pediatric lead toxicity.
At levels below 25-30 ug/dl, many of the different smaller effects reported as being
associated with lead exposure might be argued as separately not being of clear medical signi-
ficance, although each are indicative of interference by lead with normal physiological pro-
cesses. On the other hand, the collective impact of all of the observed effects (representing
potentially impaired functioning and depleted reserve capacities of many different tissues and
organs) can, at some point distinctly below 25-30 ug/dl, be seen as representing an adverse
pattern of effects worthy of avoidance. The onset of signs of detectable heme synthesis
impairment in many different organ systems at blood lead levels starting around 10-15 pg/dl,
along with indications of increasing degrees of pyrimidine metabolism interference and signs
of altered nervous system activity, might be viewed as such a point. Or, alternatively, the
collective impact of such effects might be argued as becoming sufficiently adverse to warrant
avoidance (with a margin of safety) only when the various effects come to represent marked
deviations from normal as blood lead levels exceed 20-25 ug/dl.
13-40
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The frequency of occurrence of various effects among individual affected children at
various blood lead levels may have important bearing on the ultimate resolution of the above
issue regarding the definition of blood lead levels associated with adverse health effects in
pediatric populations. The proportion of children likely affected (i.e., responders) in terms
of experiencing particular types of effects at various lead levels is also an important con-
sideration. Some information bearing on this latter point is discussed next.
13.6 DOSE-RESPONSE RELATIONSHIPS FOR LEAD EFFECTS IN HUMAN POPULATIONS
Information summarized in the preceding section dealt with the various biological effects
of lead germane to the general population and included comments about the various levels of
blood lead observed to be associated with the measurable onset of these effects in various
population groups. As indicated above, inhibition of ALA-D activity by lead occurs at virtu-
ally all blood lead levels measured in subjects residing in industrialized countries. If any
threshold for ALA-D inhibition exists, it lies somewhere below 10 pg/dl blood lead.
Elevation in erythrocyte protoporphyrin for a given blood lead level is greater in chil-
dren and women than in adult males, children being somewhat more sensitive than women. The
threshold for currently detectable EP elevation in terms of blood lead levels for children was
estimated at approximately 16-17 (jg/dl in the recent studies of Piomelli et al. (1982). In
adult males, the corresponding blood lead value is 25-30 ug/dl. Also, statistically signifi-
cant reduction in hemoglobin production occurs at a lower blood lead level in children (40
ug/dl) than in adults (50 ug/dl).
Coproporphyrin elevation in urine first occurs at a blood lead level of 40 ug/dl and this
threshold appears to apply for both children and adults. In addition, it appears that urinary
ALA shows a correlation with blood lead levels to below 40 ug/dl, but since there is no clear
agreement as to the meaning of elevated ALA-U below 40 ug/dl, this value is taken as the
threshold for pronounced excretion of ALA into urine. This value appears to apply to both
children and adults. Whether this blood lead level represents a threshold for the potential
neurotoxicity of circulating ALA cannot now be stated and requires further study.
A number of investigators have attempted to quantify more precisely dose-population re-
sponse relationships for some of the above lead effects in human populations. That is, they
have attempted to define the proportion of a population exhibiting a particular effect at a
given blood lead level. To date, such efforts at defining dose-response relationships for
lead effects have been mainly limited to the following effects of lead on heme biosynthesis:
inhibition of ALA-D activity; elevation of EP; and urinary excretion of ALA.
13-41
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Dose-population response relationships for EP in children have been analyzed in detail by
Piomelli et al. (1982) and the corresponding plot at two levels of elevation (>1 S.D., >2
S.D.) is shown in Figure 13-5 using probit analysis. It can be seen that blood lead levels in
half of the children showing EP elevations at >1 and 2 S.D.'s closely bracket the blood lead
level taken as the high end of "normal" (i.e., 30 ug/dl). Dose-response curves for adult men
and women as well as children prepared by Roels et al. (1976) are set forth in Figure 13-6.
In Figure 13-6, it may be seen that the dose-response for children remains greater across the
blood lead range studied, followed by women, then adult males.
Figure 13-7 presents dose-population response data for urinary ALA exceeding two levels
(at mean + 1 S.D. and mean + 2 S.D.), as calculated by EPA from the data of Azar et al.
(1975). The percentages of the study populations exceeding the corresponding cut-off levels
as calculated by EPA for the Azar data are set forth in Table 13-9. It should be noted that
the measurement of ALA in the Azar et al. study did not account for aminoacetone, which may
influence the results observed at the lowest blood lead levels.
99
95
90
76
O
I-
§ 10
E 6
2SD
O ^
• f
NATURAL FREQUENCY __
I
10
20
30 40
BLOOD LEAD,
60
60
70
Figure 13-5. Dose-response for elevation of EP as a
function of blood lead level using probit analysis .
Geometric mean plus 1 S.D. « 33 MQ/dl; geometric mean
plus 2 S.D. « 53
Source: Piomelli et al. (1982).
13-42
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100
•0 -
40
&
I »
ADULT FEMALES
ADULT MALES
10 20 30
BLOOD LEAD LEVEL, pg/dl
40
BO
Figure 13-6. Dose-response curve for FEP as e function
of blood lead level: in eubpopulations.
Source: Roels et et. (1976).
W10U
i-
A M
1-
M
M
40
30
20
10
I I I
I I I I 7
o MEAN +1 5.0.
AMEAN+2S.D.
MEAN ALA-U - 0.32 FOR .
BLOOD LEAD <13M/dl
I
10 20 JO 40 M CO 70
BLOOD LEAD LEVEL. pg/dl
•o
so
Figure 13-7. EPA-calculited do*e-r*sponte curve for ALA-U.
Source: Anretsl. (1975).
13-43
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TABLE 13-9. ERA-ESTIMATED PERCENTAGE OF SUBJECTS
WITH ALA-U EXCEEDING LIMITS FOR VARIOUS BLOOD LEAD LEVELS
Blood lead levels, Azar et al. (1975),
|jg/dl percent population
10 2
20 6
30 16
40 31
50 50
60 69
70 84
13.7 POPULATIONS AT RISK
A population at risk is a segment of a defined population exhibiting characteristics
associated with significantly higher probability of developing a condition, illness, or other
abnormal status. This high risk may result from either (1) greater inherent susceptibility or
(2) from exposure situations peculiar to that group. What is meant by inherent susceptibility
is a host characteristic or status that predisposes the host to a greater risk of heightened
response to an external stimulus or agent.
In regard to lead, three such populations are definable: they are preschool age children
(^6 years old), especially those living in urban settings, pregnant women, and white males
aged 40-59, although the evidence concerning this latter group is much more limited than that
for the other two. Children are such a population for both of the reasons stated above,
whereas pregnant women are at risk primarily due to the inherent susceptibility of the con-
ceptus. Also, for reasons not as yet fully understood, the limited information available
indicates that middle-aged white males appear to be differentially more at risk for mani-
festing elevations in blood pressure in response to lead exposure (see the Addendum to this
document for a complete discussion of the evidence supporting this).
13.7.1 Children as a Population at Risk
Children are developing and growing organisms exhibiting certain differences from adults
in terms of basic physiologic mechanisms, capability of coping with physiologic stress, and
their relative metabolism of lead. Also, the behavior of children frequently places them in
different relationship to sources of lead in the environment, thereby enhancing the opportu-
nity for them to absorb lead. Furthermore, the occurrence of excessive exposure often is not
13-44
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realized until serious harm is done. Young children do not readily communicate a medical his-
tory of lead exposure, the early signs of such being common to so many other disease states
that lead is frequently not recognized early on as a possible etiological factor contributing
to the manifestation of other symptoms.
13.7.1.1 Inherent Susceptibility of the Young. Discussion of the physiological vulnerability
of the young must address two discrete areas. Not only should the basic physiological differ-
ences be considered that one would expect to predispose children to a heightened vulnerability
to lead, but also the actual clinical evidence must be considered that shows such vulnerabil-
ity does indeed exist.
In Chapter 10 and Section 13.2 above, differences in relative exposure to lead and body
handling of lead for children versus adults were pinpointed throughout the text. The signifi-
cant elements of difference include the following: (1) greater intake of lead by infants and
young children into the respiratory and gastrointestinal (GI) tracts on a body weight basis
compared to adults; (2) greater absorption and retention rates of lead in children; (3) much
greater prevalence of nutrient deficiency in the case of nutrients which affect lead absorp-
tion rates from the GI tract; (4) differences in certain habits, i.e., normal hand-to-mouth
activity as well as pica, resulting in the transfer of lead-contaminated dust and dirt to the
GI tract; (5) differences in the efficiency of lead sequestration in the bones of children,
such that not only is less of the body burden of lead in bone at any given time, but the
amount present may be relatively more labile. Additional information discussed in Chapter 12
suggests that the blood-brain barrier in children is less developed, posing the risk for
greater entry of lead into the nervous system.
Hematological and neurological effects in children have been demonstrated to have lower
thresholds in terms of blood lead levels than in adults. Similarly, reduced hemoglobin pro-
duction and EP accumulation occur at relatively lower exposure levels in children than in
adults, as indexed by blood lead thresholds. With reference to neurologic effects, the onset
of encephalopathy and other injury to the nervous system appears to vary both regarding likely
lower thresholds in children for some effects and in the typical pattern of neurologic effects
presented, e.g., in encephalopathy or other CMS deficits being more common in children versus
peripheral neuropathy being more often seen in adults. Not only are the effects more acute in
children than in adults, but the neurologic sequelae are also usually much more severe in
children.
13.7.1.2 Exposure Consideration. The dietary habits of children as well as the diets them-
selves differ markedly from adults and, as a result, place children in a different relation-
ship to several sources of lead. The dominance of canned milk and processed baby food in the
diet of many young children is an important factor in assessing their exposure to lead, since
13-45
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both those foodstuffs have been shown to contain higher amounts of lead than components of the
adult diet. The importance of these lead sources is not their relationship to airborne lead
directly but, rather, their role in providing a higher baseline lead burden to which the air-
borne contribution is added.
Children ordinarily undergo a stage of development in which they exhibit normal mouthing
behavior, as manifested, for example, in the form of thumbsucking. At this time they are at
risk for picking up lead-contaminated soil and dust on their hands and hence into their mouths
where it can be absorbed.
There is, however, an abnormal extension of mouthing behavior, called pica, which occurs
in some children. Although diagnosis of this is difficult, children who exhibit this trait
have been shown to purposefully eat nonfood items. Much of the lead poisoning due to lead-
based paint is known to occur because children actively ingest chips of leaded paint.
13.7.2 Pregnant Women and the Conceptus as a Population at Risk
There are some rather inconculsive data indicating that women may in general be at some-
what higher risk to lead than men. However, pregnant women and their concepti as a subgroup
are demonstrably at higher risk. It should be noted that, in fact, it really is not the
pregnant woman per s_e who is at greatest risk but, rather, the unborn child she is carrying.
Because of obstetric complications, however, the mother herself can also be at somewhat
greater risk at the time of delivery of her child. With reference to maternal complication at
delivery, information in the literature suggests that the incidence of preterm delivery and
premature membrane rupture relates to maternal blood lead level. Further study of this rela-
tionship as well as studies relating to discrete health effects in the newborn are needed.
Vulnerability of the developing fetus to lead exposure arising from transplacental trans-
fer of maternal lead was discussed in Chapter 10. This process starts at the end of the first
trimester. Umbilical cord blood studies involving mother-infant pairs have repeatedly shown a
correlation between maternal and fetal blood lead levels.
Further suggestive evidence, cited in Chapter 12, has been advanced for prenatal lead
exposures of fetuses, possibly leading to later higher instances of postnatal mental retarda-
tion among the affected offspring. The available data are insufficient to state with any cer-
tainty that such effects occur or to determine with any precision what levels of lead exposure
might be required prior to or during pregnancy in order to produce such effects.
Studies have demonstrated that women in general, like children, tend to show a heightened
response of erythorcyte protoporphyrin levels upon exposure to lead. The exact reason for
this heightened response is not known but may relate to endocrine differences between men and
women.
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13.7.3 Middle-Aged White Males (Aged 40-59) as a Population at Risk
Recently-emerging epidemiological evidence indicates that increased blood pressure is
associated with blood lead concentrations ranging from >30-40 ug/dl down to blood lead levels
possibly as low as 7 ug/dl. This relationship appears to be particularly significant for
middle-aged white males (aged 40-59), although a considerable degree of uncertainty surrounds
the statistical analyses of the studies giving rise to this conclusion. A detailed critique
of the various analyses which have been performed on the available epidemiological studies
concerning the blood lead/blood pressure relationship, as well as a discussion of the plaus-
ible biological mechanisms underlying this relationship, are presented in Section 1 of the
Addendum to this document.
The specific magnitudes of risk obtained for serious cardiovascular outcomes in relation
to lead exposure, estimated on the basis of lead-induced blood pressure increase, depends
crucially upon the size of the coefficients estimated for the blood lead/blood pressure asso-
ciation. Given the fact that significant uncertainty exists in regard to the most appropriate
blood-lead blood-pressure coefficient(s) to use in attempting to project serious cardiovas-
cular outcomes, the further analysis of additional large-scale epidemiological data sets will
be necessary in order to resolve more precisely the quantitative relationship(s) between blood
lead and blood pressure. It is possible, however, to identify at this time the population
subgroup of middle-aged white males (aged 40-59) as being yet another group at general risk in
terms of manifesting notable health effects in response to lead exposure.
13.7.4 Description of the United States Population in Relation to Potential
Lead Exposure Risk
In this section, estimates are provided of the number of individuals in those segments of
the population which have been defined as being potentially at greatest risk for lead expo-
sures. These segments include preschool children (up to 6 years of age), especially those
living in urban settings, women of child-bearing age (defined here as ages 15-44), and white
males, aged 40-59. These data, which are presented in Table 13-10, were obtained from a
provisional report by the U.S. Bureau of the Census (1984). Data from the 1980 Census (U.S.
Bureau of the Census, 1983) indicates that approximately 61 percent of the populace lives in
urban areas (defined as central cities and urban fringe). Assuming that the 61 percent esti-
mate for total urban residents applies equally to children of preschool age, then approxi-
mately 15,495,000 children of the total listed in Table 13-10 would be expected to be at
greater risk by virtue of higher lead exposures generally associated with their living in
urban versus non-urban settings. (NOTE: The age distribution of the percentage of urban
residents may vary between SMSA's.)
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TABLE 13-10. PROVISIONAL ESTIMATE OF THE NUMBER OF INDIVIDUALS IN URBAN AND
RURAL POPULATION SEGMENTS AT GREATEST POTENTIAL RISK TO LEAD EXPOSURE
Population segment
Preschool children
Total
Women of
child-bearing age
Total
White males
Total
Actual age,
(yr)
0-4
5
6
15-19
20-24
25-29
30-34
35-39
40-44
40-44
45-49
50-54
55-59
Total number in U.S.
population
(1984)
18,453,000
3,576,000
3,374,000
25,403,000
9,019,000
10,481,000
10,869,000
10,014,000
9,040,000
7,179,000
56, 602, bOO
6,064,000
4,960,000
4,600,000
4,760,000
20,384,000
Urban
population*
11,256,000
2,181,000
2,058,000
15,495,000
5,502,000
6,393,000
6,630,000
6,109,000
5,514,000
4,379,000
34,527,000
3,699,000
3,026,000
2,806,000
2,904,000
12,435,000
*An urban/total ratio of 0.61 was used for all age groups. "Urban" includes central city
and urban fringe populations (U.S. Bureau of the Census, 1983).
Source: U.S. Bureau of the Census (1984), Table 6.
The risk encountered with exposure to lead may be compounded by nutritional deficits
(see Chapter 10). The most commonly seen of these is iron deficiency, especially in young
children less than 5 years of age (Mahaffey and Michael son, 1980). Data available from the
National Center for Health Statistics for 1976-1980 (Fulwood et al., 1982) indicate that from
8 to 22 percent of children aged 3-5 may exhibit iron deficiency, depending upon whether this
condition is defined as serum iron concentration (<40 ug/dl) or as transferrin saturation
(<16 percent), respectively. Hence, of the 22,029,000 children £5 years of age (Table 13-10),
as many as 4,846,000 would be expected to be at increased risk, depending on their exposure to
lead, due to iron deficiency.
As pointed out in Section 13.7.2, the risk to pregnant women is mainly due to risk to the
conceptus. By dividing the total number of women of child-bearing age in 1981 (56,602,000)
into the total number of live births in 1984 (3,697,000; National Center for Health Statis-
tics, 1985), it may be seen that approximately 7 percent of this segment of the population may
be at increased risk at any given time.
13-48
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As for white males, aged 40-59, defined as being at risk most notably for increased blood
pressure in association with elevated blood lead levels, approximately 20 million individuals
can be estimated to be at potential risk based on the 1980 U.S. Census data.
13.8 SUMMARY AND CONCLUSIONS
Among the most significant pieces of information and conclusions that emerge from the
present human health risk evaluation are the following:
(1) Anthropogenic activity has led to vast increases of lead input into those
environmental compartments which serve as media (e.g., air, water, food, dust,
and soil, etc.) by which significant human exposure to lead occurs. Current
blood lead concentrations of populations in industrialized societies best
reflect this impact of man's activities, with such lead levels being much
higher than those found in contemporary populations remote from industrial
activities.
(2) Emission of lead into the atmosphere, especially through leaded gasoline com-
bustion, is of major significance in terms of both the movement of lead to
other environmental compartments and the relative impact of such emissions on
the internal lead burdens in industrialized human populations. By means of
both mathematical modeling of available clinical/epidemiological data by EPA
and the isotopic tracing of lead from gasoline to the atmosphere to human blood
of exposed populations, the atmospheric lead contribution to human blood lead
levels in industrialized areas is estimated to be approximately 25-50 percent.
(3) Given this magnitude of relative contribution to human external and internal
exposure, decreases in atmospheric lead levels would then result in significant
widespread reductions in levels of lead in human blood (an outcome supported by
careful analysis of the NHANES II data). Reduction of lead in food (added in
the course of harvesting, transport, and processing) is also be expected to
produce significant widespread reductions in human blood lead levels in the
United States, as would efforts to decrease the numbers of American children
residing in housing with interior or exterior lead-based paint.
13-49
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(4) A number of adverse effects in humans and other species are clearly associated
with lead exposure and, from an historical perspective, the observed "thres-
holds" for these various effects (particularly neurological and heme biosynthe-
sis effects) continue to decline as more sophisticated experimental and clini-
cal measures are employed to detect more subtle, but still significant effects.
These include significant alterations in normal physiological functions at
blood lead levels markedly below the currently accepted 25 ug/dl "maximum safe
level" for pediatric exposures.
(5) Preceding chapters of this document demonstrate that young children are at
greatest risk for experiencing lead-induced health effects, particularly in the
urbanized, low-income segments of this pediatric population. A second group at
increased risk is pregnant women, because of exposure of the fetus to lead in
the absence of any effective biological (e.g., placental) barrier during gesta-
tion. A third group at increased risk would appear to be white males, aged
40-59, in that blood pressure elevations appear to be significantly correlated
with elevations in blood lead level in this group.
(6) Dose-population response information for heme synthesis effects, coupled with
information from various blood lead surveys, e.g., the NHANES II study, indi-
cate that large numbers of American children (especially low-income, urban
dwellers) have blood lead levels sufficiently high (in excess of 15-20 ug/dl)
that they are clearly at risk for deranged heme synthesis and, possibly, other
health effects of growing concern as lead's role as a general systemic toxicant
becomes more completely delineated.
13-50
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